Growth hormone and growth hormone releasing hormone compositions

ABSTRACT

The present invention relates to methods and compositions of growth hormone and/or growth hormone releasing hormone that promote of the release and the elevation of growth hormone when administered to animals. The present invention further relates to methods and compositions of growth hormone and/or growth hormone releasing hormone for treatment of diseases or disorders resulting from growth hormone related deficiencies. The invention also provides methods for producing novel growth hormone releasing hormone variants and their uses thereof.

[0001] The present invention relates to novel variants of growth hormonereleasing hormone (GHRH) that have enhanced resistance to enzymaticdegradation and polynucleotides encoding said GHRH variants. The presentinvention relates to methods and therapeutic compositions for thetreatment of growth hormone related deficiencies comprisingadministrating to humans, companion animals, livestock or poultry,plasmid compositions comprising polynucleotides encoding GHRH orvariants thereof, alone or in combination with polynucleotides encodinggrowth hormone or modified growth hormone. The present invention furtherrelates to methods and compositions that promote the release andexpression of growth hormone in order to enhance the growth andperformance of companion animals, livestock or poultry comprising theadministration plasmid compositions encoding GHRH variants, GHRH ormodified GHRH, and/or GH or modified GH.

BACKGROUND OF THE INVENTION

[0002] Growth hormone-releasing hormone (“GHRH”) is a peptide hormonesecreted from the hypothalamus. Following secretion, GHRH enters theportal circulation connecting the hypothalamus to pituitary gland. GHRHthen interacts with its receptors on the pituitary gland and induces therelease of growth hormone (“GH”). GH secreted from the pituitary glandenters the general circulation and from there it reaches various organsand tissues of the body where it interacts with specific receptors andinduces a wide range of developmental effects.

[0003] GHRH peptides have been isolated and characterized from severalspecies including humans, porcine, ovine and bovine. In each of thesespecies, GHRH is a small polypeptide consisting of 44 amino acids(GHRH(1-44)-NH₂). However, it has been also shown that smallerfragments, most notably those consisting of the first (amino terminal)29 amino acids (referred to as GHRH1-29 fragment) retain the sameintrinsic biological activity as the full length parent molecule.

[0004] GHRH is synthesized as a precursor polypeptide consisting of 107or 108 amino acids depending on the species. Following synthesis, theprecursor GHRH polypeptide undergoes sequential processing. First, the31 amino acid signal peptide (Met⁻³⁰ to Arg⁰) of the GHRH precursorpolypeptide is cleaved (Smith et al., 1992, Biotechnology 10:315-319).Subsequently, the GHRH precursor polypeptide is cleaved at position46-47 and at position 45-46 by a trypsin-like endopeptidase and acarboxypeptidase, respectively, resulting in generation of GHRH(1-45)-OHand a 30 amino acid peptide (amino acids 77-107) designated GCTP (Brar,A. K. et al., 1991, Endocrinology 129: 3274-3280). The GHRH(1-45)-OHpolypeptide is further processed by peptidyl glycine α-amidatingmonooxygenase (“PAM”), which transfers an amide group from Gly⁴⁵ toLeu⁴⁴ and results in the formation of GHRH(1-44)-NH₂, the full lengthform of GHRH (Brar, A. K. et al., 1991, Endocrinology 129: 3274-3280).The GCTP is also undergoes processing by PAM, which results in thetransfer of an amide group from Gly⁷⁷ to Gln⁷⁶. Although the role ofGHRH(1-44)-NH₂ in inducing the release of GH is well established, therole of the GCTP peptide is less clear. One report has implicated theGCTP peptide in the control of feeding behavior (Arase, K. et al., 1987,Endocrinology 121:1960-1965).

[0005] GH has been identified and its gene cloned from many speciesincluding human, porcine, bovine, and equine. Unlike GHRH, there existsnatural variants of GH within a given species. For example, bovine GH isreleased from the pituitary gland in one of four variants which differfrom one another by one or more amino acids and some studies suggestthat these variants differ in their potency (e.g., in terms of theirability to increase milk yield). Several studies have also identifiedamino acid substitutions that lead to an increase in the affinity of GHto its receptors and/or enhanced stability to enzymatic degradation.Studies have also shown that immunization against specific peptides fromGH (e.g., a peptide consisting of amino acids 35 to 53 of GH) leads toproduction of antibodies that bind growth hormone and increase theefficacy of GH treatment, presumably because the antibodies delay theclearance of GH from circulation, thus, increasing half-life of GH,and/or protect GH from proteolytic degradation (Bomford, F. and Aston,P., 1990, Endocrinology 125:31-38).

[0006] Significant research efforts have focused on the structuralattributes of GH and GHRH, as well as their biological and developmentalactivities. A number of groups have attempted to exploit GH and GHRH ina manner that could provide important therapeutic and economic benefitsas a result of their use in humans and animals. For example, thetraditional treatment of GH-deficient children has been theadministration of growth hormone isolated from human pituitary glands,however these preparations are no longer available in the United Statesdue to virus-contaminated samples (Vance, 1990, Clin. Chem 36/3:415-420). Recombinantly expressed and purified GH have been shown tohave some benefits in treating GH-deficient children, however thecombination of recombinantly expressed GH and GHRH in the treatment ofGH-deficient children has provided conflicting results. (Vance, supra).Further, purified GH and GHRH must be administered at very highquantities to be effective as the exposure of these polypeptides toserum results in their rapid degradation to a polypeptide which exhibitsconsiderably different biological and pharmacokinetic properties.(Fronman et al., 1989, J. Clin. Invest. 83:1533-1540).

[0007] Other studies have shown that GH or GHRH administered as purifiedpolypeptides have significant impact on animal growth (muscle and bonegrowth), average daily gain, milk production, feed efficiency (the ratioof feed consumed to body weight gain), adipose tissue accretion andothers. For example, it has been shown that daily administration ofmaximally effective doses of GH administered to growing pigs for 33-77days can increase average daily gain −10-20%, improves feed efficiency13-33%, decrease adipose tissue accretion by as much as 70%, andstimulates protein deposition (muscle growth) by as much as 62%.(Etherton et al.,1998, Physiological Reviews 78:745-761). Furthermore,when GH was administered to dairy cows, milk yields were increased by10-15% (□4-6kg/day) (Etherton et al.,1998, Physiological Reviews78:745-761).

[0008] A major impediment to fulfilling the therapeutic and economicpotential of GHRH peptides is their susceptibility to cleavage (andsubsequent conversion to inactive forms) by specific tissue and plasmaproteolytic enzymes; most notably dipeptidylpeplidase IV (“DPPIV”). Anumber of researchers have focused on manipulating GHRH in order todevelop compounds with significant therapeutic potential. Consequently,a wide variety of synthetic GHRH peptide analogues have been produced.They consist of GHRH polypeptides in which one or more amino acids havebeen chemically modified or replaced with other L- or D-amino acids.These modifications or substitutions are designed to yield analogueswith biological properties superior to those of the parent molecule interms of potency, stability and resistance to chemical and enzymaticdegradation. However, these chemically modified polypeptides are noteasily or efficiently produced in a suitable form to be administered tohumans or animals.

[0009] In spite of the significant therapeutic and economic benefits ofGH or GHRH alluded to above, exogenous supplementation of animals withGH or GHRH proteins have not been widely adopted as a component ofroutine management practices to enhance the quality of meat from animalsand/or enhance the productivity of livestock. This is because in orderto get these benefits, animals have to be repeatedly administered GH orGHRH polypeptides (often daily, but typically in a slow releaseformulation given every 7-10 days). (Etherton, T. D., 1997, NatureBiotechnology 15:12⁴8) This situation is labor intensive, timeconsuming, expensive, and does not fit current management practiceswhere animals are reared in large numbers and are handled veryinfrequently, it is apparent therefore that in order to realize thetherapeutic and economic benefits of GH and/or GHRH administration, muchimproved formulations for delivery of these hormones must be developedto overcome the current limitations of their use; namely the need forrepeated administration.

SUMMARY OF THE INVENTION

[0010] The present invention relates to novel variants of GHRH that haveenhanced resistance to enzymatic degradation and polynucleotidesencoding said variants. The present invention also relates topharmaceutical formulations comprising polynucleotide sequences encodingGHRH variants alone or in combination with polynucleotide sequencesencoding GHRH, modified GHRH, GH and/or modified GH. The presentinvention also relates to pharmaceutical formulations comprising GHRHvariant peptides alone or in combination with GHRH polypeptides,modified GHRH polypeptides, GH polypeptides and/or modified GHpolypeptides. The present invention further relates to pharmaceuticalformulations comprising canine or feline GHRH peptides alone or incombination with GHRH variant polypeptides, modified GHRH polypeptides,GH polypeptides and/or modified GH polypeptides.

[0011] The present invention relates to therapeutic methods andcompositions for the treatment of growth hormone related deficienciescomprising growth hormone (“GH”) and/or growth hormone-releasing hormone(“GHRH”) in human, companion animals, livestock and poultry. Theinvention also relates to methods for the improvement in the health ofhumans, companion animals, livestock and poultry. The invention alsorelates to methods for the treatment of obesity and frailty of companionanimals. The invention further relates to methods for the enhancement ofthe growth and performance of companion livestock and poultry. Themethods of the present invention comprise pharmaceutical compositionswhich enhance the expression of growth hormone or promote the release ofgrowth hormone or both when administered to humans, companion animals,livestock or poultry. According to the present invention, the term“GHRH” relates to the full length wildtype form of GHRH which is 44amino acids (aa) or a precursor form of GHRH. In accordance with thepresent invention, the term “modified GHRH” refers to any amino terminalpolypeptide fragment of GHRH from 29 amino acids to 107 or 108 aminoacids in length and any mutant of GHRH, including additions, deletionsor substitutions at the nucleotide or amino acid level, which retains atleast the level of activity of wildtype GHRH, that is, the ability toinduce GH gene transcription at levels comparable to wildtype GHRH.

[0012] In accordance with the present invention, the term “GHRH variant”relates to a GHRH polypeptide to which one or more amino acids have beenattached to the carboxy or amino terminus of the polypeptide, or awildtype GHRH polypeptide that contains a substitution of one or moreamino acids, so that the GHRH variant retains at least equal or enhancedwildtype GHRH activity and has enhanced resistance to enzymaticdegradation relative to the wildtype GHRH. In accordance with thepresent invention, wildtype GHRH activity is measured by its ability toinduce GH gene transcription. In accordance with the present invention,resistance to enzymatic degradation is determined by the ability of thepolypeptide to resist degradation caused by dipeptidylpeptidase type IV.

[0013] According to the present invention, the term “GH” refers to thefull length wildtype form of GH, which is 191 amino acids, and “modifiedGH” refers to any fragment of GH and any mutant including additions,deletions or substitutions at the nucleotide or amino acid level, whichretains at least the level of wildtype activity of GH, that is, theability to induce insulin growth factor (IGF) gene transcription atlevels comparable to wildtype GH, or mimic the anti-adipogenic andlipolytic effects of GH.

DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a map of the pGHRH4 construct (SEQ ID No.47).

[0015]FIG. 2 is a map of the pGHRH1-44SK construct (SEQ ID No.48).

[0016]FIG. 3 is a map of the pGHRH1-44WTSK685 construct (SEQ ID No. 49).

[0017]FIG. 4 is a map of the pGHRH1-44WTSK2014 construct (SEQ ID No.50).

[0018]FIG. 5 is a graph depicting the GHRH expression levels detected insupernatants from pGHRH4 transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to novel variants of GHRH that haveenhanced resistance to enzymatic degradation and polynucleotidesencoding said GHRH variants. The present invention relates topharmaceutical compositions which promote the release and/or expressionof GH. In particular, the pharmaceutical compositions of the presentinvention comprise polynucleotide sequences encoding GHRH variants aloneor in combination with polynucleotide sequences encoding GHRH, modifiedGHRH, GH and/or modified GH or any combination thereof. In anotherembodiment, the pharmaceutical compositions of the present inventioncomprise GHRH variant polypeptides alone or in combination with GHRHpolypeptides, GHRH modified polypeptides, GH polypeptides or GH modifiedpolypeptides or any combination thereof.

[0020] The present invention relates to methods of treating disordersrelated to GH related deficiencies in humans, companion animals,livestock and poultry, comprising administering pharmaceuticalformulations which enhance GH expression and/or release. The presentinvention further relates to methods of treating livestock and poultryin order to enhance growth and performance comprising administeringpharmaceutical formulations which enhance GH expression and/or release.

[0021] The pharmaceutical formulations to be administered in accordancewith the methods of the present invention encompass plasmid compositionscomprising (a) polynucleotide sequences encoding GHRH variants; (b)polynucleotide sequences encoding GHRH or modified GHRH; (c)polynucleotide sequences encoding GH or modified GH; or any combinationthereof, wherein the polynucleotide sequences are operably linked to apromoter or regulatory element, preferably one that is transcriptionallyactive in muscle tissue. The pharmaceutical formulations to beadministered in accordance with the methods of the present inventionalso include: i) plasmid compositions comprising polynucleotidesencoding for GH or modified or variant GHRH gene; ii) plasmidcompositions comprising polynucleotides encoding for both GH and GHRHgenes; iii) plasmid compositions comprising polynucleotides encoding fora GHRH gene, a GH gene or a gene encoding a fusion protein consisting ofa peptide from GH and a carrier protein for induction of GHpotency-enhancing antibodies; (iv) recombinant proteins, peptides,fragments or derivatives thereof comprising canine GHRH or feline GHRH;(v) recombinant proteins, peptides, fragments or derivatives thereof ofthe GHRH variants of the present invention; and (vi) recombinant fusionproteins, peptides, fragments or derivatives thereof comprising GH andGHRH.

[0022] In one embodiment, the pharmaceutical compositions of the presentinvention comprise polynucleotide sequences encoding canine or felineGHRH alone or in combination with polynucleotide sequences encoding GHRHvariant, modified GHRH, GH and/or modified GH or any combinationthereof. In another embodiment, the pharmaceutical compositions of thepresent invention comprise canine or feline GHRH polypeptides alone orin combination with GHRH variant polypeptides, modified GHRH, GHpolypeptides or GH modified polypeptides or any combination thereof. Thepharmaceutical compositions of the present invention are in suitableformulation to be administered to humans, companion animals, livestockor poultry for the treatment of growth hormone related deficiencies orthe enhancement of growth and performance of livestock and poultry. Thepharmaceutical compositions of the present invention are also insuitable formulation for the treatment of obesity and frailty ofcompanion animals or the improvement in the health of humans, companionanimals, livestock, and poultry.

[0023] The present invention relates to therapeutic methods andcompositions for the treatment of growth hormone related deficienciescomprising growth hormone (“GH”); modified GH; growth hormone releasinghormone (“GHRH”); GHRH variants; modified GHRH or any combinationthereof. The therapeutic compositions of the invention are administeredto animals, preferably to mammals, more preferably to companion animals(e.g., dogs, cats and horses), livestock (e.g., cows and pigs) andpoultry (e.g., chickens and turkeys), and most preferably to humans. Theinvention also relates to methods and compositions for the enhancementof the growth and performance of animals, more preferably mammals, andmost preferably livestock (e.g., cows and pigs) and poultry (e.g.,chickens and turkeys) with the proviso that such compositions are not tobe administered to mice, rats, rodents, guinea pigs, or rabbits. Theinvention also relates to methods and compositions for the treatment ofobesity and frailty of animals, preferably to mammals, more preferablyto companion animals (e.g., dogs, cats and horses). The inventionfurther relates to methods and compositions for the improvement in thehealth of animals, preferably to mammals, more preferably to companionanimals (e.g., dogs, cats and horses), livestock (e.g., cows and pigs)and poultry (e.g., chickens and turkeys), and most preferably to humans.

[0024] The present invention is based in part of the discovery ofrecombinantly engineered GHRH variants which retain at least the levelof activity wildtype GHRH, that is the ability to induce GH genetranscription at levels comparable to wildtype GHRH, and which haveenhanced resistance to enzymatic degradation relative to the wildtypeGHRH. The GHRH variants of the present invention may be recombinantlyexpressed at high levels in host cells and easily isolated and purifiedin a form suitable for administration to humans and animals. Thus, theGHRH variants of the present invention may be efficiently produced andisolated at high levels as opposed to modified GHRH polypeptides in theart which are modified using traditional chemistry methods to introducemodifications in the native GHRH sequence.

[0025] In one embodiment, a GHRH variant of the present inventioncomprises the addition of one amino acid, preferably a hydrophobicresidue and more preferably a tyrosine residue, to the amino terminus(position 1) of GHRH. In another embodiment, a GHRH variant comprisesthe addition of two amino acids, wherein the second amino acid is notproline or alanine, to the amino terminus (position 1) of GHRH. Inanother embodiment, a GHRH variant comprises the addition of three aminoacids, wherein the second amino acid is proline or alanine, to the aminoterminus (position 1) of GHRH. In another embodiment, a GHRH variantcomprises the addition of more than three amino acids to the aminoterminus (position 1) of GHRH, wherein the addition does not interferewith the functional activity of GHRH, that is, the ability of GHRH toinduce GH gene transcription. In a preferred embodiment of the presentinvention, a GHRH variant comprises the addition of a tripeptide to theamino terminus, wherein the tripeptide is diprotin A or diprotin B or apeptide with a structure analogous to diprotin A or diprotin B. In yetanother embodiment, a GHRH variant comprises the addition of glycine andarginine at the carboxy-terminus. This addition results in the amidationof GHRH; the glycine and arginine is cleaved off and the last amino acidbefore the added glycine is amidated.

[0026] In one embodiment, a GHRH variant comprises any of the amino acidadditions described above and the substitution of glycine with alanineat residue 15. In another embodiment, a GHRH variant comprises any ofthe amino acid additions described above and the substitution of leucinewith alanine at residue 22. In another embodiment, a GHRH variantcomprises any of the amino acid additions described above and thesubstitutions of glycine with alanine at residue 15 and leucine withalanine at residue 22. In another embodiment, a GHRH variant comprisesany of the amino acid additions at the amino terminus and the amino acidadditions at the carboxy-terminus. In another embodiment, a GHRH variantcomprises any of the amino acid additions at the amino terminusdescribed above, the amino acid additions at the carboxy-terminusdescribed above, and the substitution of glycine to alanine at residue15. In another embodiment, a GHRH variant comprises any of the aminoacid additions at the amino terminus described above, the amino acidadditions at the carboxy-terminus described above, and the substitutionof leucine to alanine at residue 22. In yet another embodiment, a GHRHvariant comprises any of the amino acid additions at the amino terminusdescribed above, the amino acid additions at the carboxy-terminusdescribed above, and the substitutions of glycine to alanine at residue15 and leucine to alanine at residue 22. The term “GHRH precursorvariant” as used herein refers to a precursor form of the full lengthwildtype GHRH polypeptide to which one or more amino acids have beenattached to the amino terminus of the polypeptide and/or contains asubstitution of one or more amino acids, so that the GHRH precursorvariant retains at least equal or enhanced wildtype GHRH activity andhas enhanced resistance to enzymatic degradation relative to wildtypeGHRH. In one embodiment of the present invention, a GHRH precursorvariant comprises the amino acid additions and/or the amino acidsubstitutions described above. The present invention also encompasses afusion variant comprising any of the GHRH variants described above andGH or modified GH. The term “fusion variant” as used herein refers to afusion protein comprising GHRH variants or modified GHRH and GH ormodified GH. In one embodiment of the present invention, a fusionvariant comprises any of the GHRH variants described above, whichconsist of amino acid additions at the amino terminus and/or amino acidsubstitutions, and GH or modified GH.

[0027] The modifications and/or substitutions of GHRH described hereinare made to GHRH polypeptides which retain the biological activity atleast equal to the full length wildtype GHRH, preferably a precursorform of GHRH, more preferably the sequence of GHRH consisting of about29 amino acids to about 44 amino acids. The polynucleotide sequencesencoding the GHRH variants described herein are also within the scope ofthe present invention. The present invention provides that thepolypeptides are encoded by the nucleic acid fragments of the presentinvention or by degenerate variants of the nucleic acid fragments of thepresent invention. By “degenerate variant” is intended nucleotidefragments which differ from a nucleic acid fragment of the presentinvention (e.g., an ORF) by nucleotide sequence but, due to thedegeneracy of the genetic code, encode an identical polypeptidesequence. Preferred nucleic acid fragments of the present invention arethe ORFs that encode proteins. The present invention encompasses GHRHvariants encoded by the polynucleotide sequence any species.

[0028] The present invention encompasses polynucleotide sequencesencoding precursor forms of GHRH, full length wildtype GHRH, modifiedGHRH, GHRH variants, and fragments of GHRH from 29 amino acids to 44amino acids in length for any species, which retain at least theactivity of wildtype GHRH. For example, the polynucleotide sequencesencoding human, swine, and bovine growth hormone releasing hormonedisclosed in Genbank accession number SEG_HSGHRH, accession numberU90275, and accession number U29611, respectively, are incorporatedherein by reference. The present invention also encompassespolynucleotide sequences encoding GHRH polypeptides disclosed for anyspecies (e.g., the polynucleotide sequence encoding the human GHRHprecursor polypeptide disclosed in Genbank accession number P01286 isincorporated herein by reference). The present invention furtherencompasses polynucleotide sequences encoding full length wildtype GH ormodified GH for any species. For example, the polynucleotide sequencesencoding human, swine, and bovine growth hormone disclosed in Genbankaccession number J03071, accession number U19787, and accession numberE00293, respectively, are incorporated herein by reference. The presentinvention also encompasses polynucleotide sequences encoding GHpolypeptides disclosed for any species (e.g., the polynucleotidesequence encoding the bovine GH polypeptide disclosed in Genbankaccession number STBO is incorporated herein by reference).

[0029] The polynucleotide sequence encoding GHRH, modified GHRH or GHRHvariants can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The polynucleotidesequence encoding GH or modified GH can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native GH or native GHRH genes or its flankingregions. A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In one embodiment, the wildtype or modified human GH gene is expressed.In another embodiment, the wildtype, modified or variant human GHRH isexpressed. In yet another embodiment, the wildtype or modified human GHand the wildtype, modified or variant human GHRH gene are expressed.

[0030] Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene, comprising GH or modified GH and GHRH,modified GHRH or GHRH variants, consisting of appropriatetranscriptional and translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of the nucleic acid sequence encoding GH or modified GH maybe regulated by a second nucleic acid sequence so that the GH ormodified GH is expressed in a host transformed with the recombinant DNAmolecule. Expression of the nucleic acid sequence encoding GHRH,modified GHRH or GHRH variant may be regulated by a second nucleic acidsequence so that the GHRH modified GHRH or GHRH variant is expressed ina host transformed with the recombinant DNA molecule. For example,expression of GH or GHRH may be controlled by any promoter or enhancerelement known in the art. Promoters which may be used to control GHand/or GHRH gene expression include, but are not limited to, theCytomeglovirus (CMV) immediate early promoter region, the SV40 earlypromoter region (Bernoist and Chambon, 1981, Nature 290:304-310), thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445),the regulatory sequences of the metallothionein gene (Brinster et al.,1982, Nature 296:3942); prokaryotic expression vectors such as theβ-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad.Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc.Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins fromrecombinant bacteria” in Scientific American, 1980, 242:74-94; plantexpression vectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant Biol.50:399409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), swine alpha-skeletal actin control region which isactive in muscle (Reecy, M. et al., 1998, Animal Biotechnology9:101-120) and gonadotropic releasing hormone gene control region whichis active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

[0031] In a specific embodiment, a vector is used that comprises apromoter operably linked to GH- or modified GH-encoding nucleic acid,one or more origins of replication, and, optionally, one or moreselectable markers (e.g., an antibiotic resistance gene). In anotherembodiment, a vector is used that comprises a promoter operably linkedto GHRH-, modified GHRH- or GHRH variant-encoding nucleic acid, one ormore origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene). In yet anotherembodiment, a vector is used that comprises a promoter operably linkedto GH or modified GH and GHRH, modified GHRH or GHRH variant-encodingnucleic acids, one or more origins of replication, and, optionally, oneor more selectable markers (e.g., an antibiotic resistance gene).

[0032] Expression vectors containing gene inserts can be identified bythree general approaches: (a) nucleic acid hybridization, (b) presenceor absence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of the GH- or modifiedGH-encoding polynucleotides and GHRH-, modified GHRH- or GHRHvariant-encoding polynucleotides inserted in an expression vector(s) canbe detected by nucleic acid hybridization using probes comprisingsequences that are homologous to the inserted genes. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of the gene(s) in the vector(s). For example, ifthe GH gene is inserted within the marker gene sequence of the vector,recombinants containing the GH gene insert can be identified by theabsence of the marker gene function. In the third approach, recombinantexpression vectors can be identified by assaying the gene productexpressed by the recombinant. Such assays can be based, for example, onthe physical or functional properties of the GH and GHRH in in vitroassay systems, e.g., binding of GH with anti-GH antibody or binding ofGHRH with anti-GHRH antibody.

[0033] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

[0034] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered may be controlled. Furthermore,different host cells have characteristic and specific mechanisms for thetranslational and post-translational processing and modification (e.g.,glycosylation, phosphorylation of proteins). Appropriate cell lines orhost systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an unglycosylated core proteinproduct. Expression in yeast will produce a glycosylated product.Expression in mammalian cells can be used to ensure “native”glycosylation of a heterologous protein. Furthermore, differentvector/host expression systems may effect processing reactions todifferent extents.

[0035] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the differentially expressed or pathway gene protein may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express the differentially expressed orpathway gene protein. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the differentially expressed or pathway geneprotein.

[0036] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genescan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30:147) genes.

[0037] The present invention provides for the treatment or prevention ofGH associated diseases or disorders, including those disorderscharacterized by GH deficiency, comprising the administration of apharmaceutical formulation to a human, companion animal, livestock orpoultry which enhances GH expression and/or release. The presentinvention also provides for methods and protocols to enhance the growthand performance of livestock and poultry, comprising the administrationof a pharmaceutical formulation to a companion animal, livestock orpoultry which enhances GH expression and/or release. The presentinvention also provides for the treatment of obesity and frailty,comprising the administration of a pharmaceutical formulation to acompanion animal which enhances or modulates GH expression and/orrelease. The present invention further provides for methods for theimprovement in health, comprising the administration of a pharmaceuticalformulation to a human, companion animal, livestock or poultry whichenhances GH expression and/or release. In accordance with the invention,methods of the present invention encompass the administration ofpharmaceutical formulations comprising: (a) polynucleotide sequencesencoding GHRH variants alone or in combination with polynucleotidesequences encoding GHRH, modified GHRH, GH, modified GH or anycombination thereof, wherein the polynucleotide sequences are operablylinked to a promoter or regulatory element, preferably one that istranscriptionally active in muscle tissue; (b) polynucleotide sequencesencoding canine or feline GHRH alone or in combination withpolynucleotide sequences encoding GHRH variants, modified GHRH, GH,modified GH or any combination thereof, wherein the polynucleotidesequences are operably linked to a promoter or regulatory element,preferably one that is transcriptionally active in muscle tissue; (c)polynucleotide sequences encoding GHRH, modified GHRH, GH, modified GHor any combination thereof wherein the polynucleotide sequences areoperably linked to a promoter or regulatory element, preferably one thatis transcriptionally active in muscle tissue; (c) variant GHRHpolypeptides alone, expressed as a fusion protein, or in combinationwith GHRH, modified GHRH, GH or modified GH polypeptides or anycombination thereof; or (d) canine or feline GHRH polypeptides alone,expressed as a fusion protein, or in combination with GHRH variants,modified GHRH, GH or modified GH polypeptides or any combinationthereof.

[0038] Generally, administration of products of a species origin orspecies reactivity (in the case of antibodies) that is the same speciesas that of the patient is preferred. Thus, in a preferred embodiment,human GH and/or GHRH genes, gene fragments or derivatives thereof areadministered to a human patient for therapy or prophylaxis.

[0039] In a specific embodiment, nucleic acids comprising sequencesencoding GH and/or GHRH or functional derivatives thereof, areadministered to promote the release and/or elevation of growth hormone,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediate a therapeutic effect by promoting thefunction of GH.

[0040] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0041] For general reviews of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

[0042] In a preferred aspect, the compound comprises nucleic acidsequences encoding GH or modified GH and GHRH, modified GHRH or GHRHvariants, said nucleic acid sequences being part of expression vectorsthat express GH or modified GH and GHRH, modified GHRH or GHRH variantsin a suitable host. In particular, such nucleic acid sequences havepromoters operably linked to the GH or modified GH and GHRH, modifiedGHRH or GHRH variants coding regions, said promoters being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the GH or modifiedGH and GHRH, modified GHRH or GHRH variants coding sequences and anyother desired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the GH or modified GH and GHRH, modifiedGHRH or GHRH variants nucleic acids (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; Zijistra et al., 1989, Nature342:435-438).

[0043] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0044] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

[0045] In a specific embodiment, viral vectors that contain nucleic acidsequences encoding GH or modified GH and/or GHRH, modified GHRH or GHRHvariants are used. For example, a retroviral vector can be used (seeMiller et al., 1993, Meth. Enzymol. 217:581-599). These retroviralvectors have been to delete retroviral sequences that are not necessaryfor packaging of the viral genome and integration into host cell DNA.The nucleic acid sequences encoding the GH or modified GH and GHRH,modified GHRH or GHRH variants to be used in gene therapy are clonedinto one-or more vectors, which facilitates delivery of the gene into apatient. More detail about retroviral vectors can be found in Boesen etal., 1994, Biotherapy 6:291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.3:110-114.

[0046] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used. Adeno-associatedvirus (AAV) has also been proposed for use in gene therapy (Walsh etal., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No.5,436,146).

[0047] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0048] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0049] The resulting recombinant cells can be delivered to a subject byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, subject=s state, etc., and can be determined by oneskilled in the art.

[0050] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0051] In a preferred embodiment, the cell used for gene therapy isautologous to the subject.

[0052] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding GH or modified GH and/or GHRH,modified GHRH or GHRH variants are introduced into the cells such thatthey are expressible by the cells or their progeny, and the recombinantcells are then administered in vivo for therapeutic effect. In aspecific embodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see e.g. PCT Publication WO 94/08598, dated Apr. 28, 1994;Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. CellBio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

[0053] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0054] The polypeptides of the invention include polypeptides whichcomprise the amino acid sequence of canine or feline GHRH. Thepolypeptides of the invention also include polypeptides which comprisethe amino acid sequence of a GHRH variant of the present invention. Thepolypeptides of the invention further include polypeptides whichcomprise the amino acid sequence of GH or modified and GHRH or modifiedGHRH. Protein compositions of the present invention may further comprisean acceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

[0055] The invention also relates to methods for producing a polypeptidecomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing apolypeptide in which a host cell containing a suitable expression vectorthat includes a polynucleotide of the invention is cultured underconditions that allow expression of the encoded polypeptide. Thepolypeptide can be recovered from the culture, conveniently from theculture medium, and further purified.

[0056] The present invention further provides isolated polypeptidesencoded by the nucleic acid fragments of the present invention or bydegenerate variants of the nucleic acid fragments of the presentinvention. By “degenerate variant is intended nucleotide fragments whichdiffer from a nucleic acid fragment of the present invention (e.g., anORF) by nucleotide sequence but, due to the degeneracy of the geneticcode, encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins. Avariety of methodologies known in the art can be utilized to obtain anyone of the isolated polypeptides or proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides.Fragments are useful, for example, in generating antibodies against thenative polypeptide. In an alternative method, the polypeptide or proteinis purified from bacterial cells which naturally produce the polypeptideor protein. One skilled in the art can readily follow known methods forisolating polypeptides and proteins in order to obtain one of theisolated polypeptides or proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology.

[0057] The polypeptides and proteins of the present invention canalternatively be purified from cells which have been altered to expressthe desired polypeptide or protein. As used herein, a cell is said to bealtered to express a desired polypeptide or protein when the cell,through genetic manipulation, is made to produce a polypeptide orprotein which it normally does not produce or which the cell normallyproduces at a lower level. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention. The purified polypeptides can be used in in vitrobinding assays which are well known in the art to identify moleculeswhich bind to the polypeptides. These molecules include but are notlimited to, for e.g., small molecules, molecules from combinatoriallibraries, antibodies or other proteins.

[0058] The protein of the invention may also be expressed as a productof transgenic animals, e.g., as a component of the milk of transgeniccows, goats, pigs, or sheep which are characterized by somatic or germcells containing a nucleotide sequence encoding the protein.

[0059] The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon-therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

[0060] The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods arewell known in the art, as described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), incorporatedherein by reference. As used herein, an insect cell capable ofexpressing a polynucleotide of the present invention is “transformed.”

[0061] The protein of the invention may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant protein. The resulting expressed protein may then bepurified from such culture (i.e., from culture medium or cell extracts)using known purification processes, such as gel filtration and ionexchange chromatography. The purification of the protein may alsoinclude an affinity column containing agents which will bind to theprotein; one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl.RTM. or Cibacrom blue 3GASepharose.RTM.; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

[0062] Alternatively, the protein of the invention may also be expressedin a form which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and In Vitrogen, respectively. The protein can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (“Flag”) iscommercially available from Kodak (New Haven, Conn.).

[0063] Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

[0064] The compounds of the invention are preferably tested in vitro,and then in vivo for the desired therapeutic or prophylactic activity,prior to use in humans. For example, in vitro assays which can be usedto determine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0065] The expression of GH or modified GH and GHRH, modified GHRH orGHRH variants can be assayed by the immunoassays, gel electrophoresisfollowed by visualization, or any other method known to those skilled inthe art.

[0066] In various specific embodiments, in vitro assays can be carriedout with representative cells of cell types involved in a patient'sdisorder, to determine if a compound has a desired effect upon such celltypes. In accordance with the present invention, the functional activityof GHRH can be measured by its ability to induce GH gene transcriptionin vitro. In accordance with the present invention, the functionalactivity of GHRH can be measured by its ability to induce IGF genetranscription in vitro.

[0067] Compounds for use in therapy can be tested in suitable animalmodel systems prior to testing in humans, including but not limited topigs, chicken, cows or monkeys.

[0068] The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a compound of theinvention. In a preferred aspect, the compound is substantially purified(e.g., substantially free from substances that limit its effect orproduce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is-preferably a mammal, and mostpreferably human. In a specific embodiment, a non-human mammal is thesubject.

[0069] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid are described above;additional appropriate formulations and routes of administration can beselected from among those described herein below.

[0070] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intratumoral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceuticalcompositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0071] In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

[0072] In another embodiment, the compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

[0073] In yet another embodiment, the compound can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

[0074] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0075] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0076] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0077] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0078] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree amino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0079] The amount of the compound of the invention which will beeffective in the treatment of cancer can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-500 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

[0080] Suppositories generally contain active ingredient in the range of0.5% to 10% by weight; oral formulations preferably contain 10% to 95%active ingredient.

[0081] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

EXAMPLES Cloning of Canine and Feline GHRH

[0082] 1. Cloning of Canine GHRH

[0083] In order to clone the canine GHRH, the canine genomic library(Clonetech Lab) is screened with the radioactively labeled fragmentencoding the porcine GHRH (SEQ ID No. 1) following protocols known toone of ordinary skill in the art. The fragment encoding the porcine GHRH(SEQ ID No. 1) is labeled using commercially available DNA labeling kitsas recommended by the manufacturer. Clones identified that contain thegene coding for the canine precursor GHRH are isolated and sequenced bymethods known to one of ordinary skill in the art. The sequence resultsobtained from sequencing both DNA strands of a clone is compared withsequences of known GHRH species and the canine GHRH is subcloned intoappropriate plasmid vectors (e.g., pVR1012; Vical, San Diego, Calif.)according to protocols known to one of ordinary skill in the art.

[0084] 2. Cloning of Feline GHRH

[0085] In order to clone the feline GHRH, the feline genomic library(Clonetech Lab) is screened with the radioactively labeled fragmentencoding the porcine GHRH (SEQ ID No. 1) following protocols known toone of ordinary skill in the art. The fragment encoding the porcine GHRH(SEQ ID No. 1) is labeled using commercially available DNA labeling kitsas recommended by the manufacturer. Clones identified that contain thegene coding for the feline precursor GHRH are isolated and sequenced bymethods known to one of ordinary skill in the art. The sequence resultsobtained from sequencing both DNA strands of a clone is compared withsequences of known GHRH species and the feline GHRH is subcloned intoappropriate plasmid vectors (e.g., pVR1012; Vical, San Diego, Calif.)according to protocols known to one of ordinary skill in the art.

Synthesis of GHRH Constructs

[0086] 3. pGHRH-4 (pGHRH1-44WTCMV)

[0087] In order to construct a plasmid containing the gene that codesfor the natural porcine GHRH polypeptide and to have the latter secretedinto the blood circulation when such plasmids are injected into animals,primers designated GHRH-1 (SEQ ID No. 61), GHRH-2 (SEQ ID No. 62),GHRH-4 (SEQ ID No. 66), and GHRH-7 (SEQ ID No. 67) were synthesized. Theprimers were used in reverse transcription polymerase chain reactions(RT-PCRs) to amplify the human GHRH signal sequence from human mRNA andthe porcine GHRH protein sequence from porcine mRNA. The resultingPCR-amplified human GHRH signal sequence and porcine GHRH proteinsequence were digested with Bgl II and Bam HI, respectively. Then thefragments were ligated together and cloned into the Bam HI site of theplasmid pVR1012 (Vical, San Diego, Calif.) to produce a plasmiddesignated pGHRH-4 (FIG. 1). The expression of the GHRH oligonucleotide(SEQ ID Nos. 1 and 2), which encodes a 75 amino acid polypeptidecomprising the porcine GHRH protein sequence (44 amino acids) precededby the signal sequence from human GHRH protein (31 amino acids), isdriven by Cytomegalo-virus immediate early (CMV IE) promoter/enhancerelement.

[0088] 4. pGHRH1-44WTSK685:

[0089] A plasmid containing the polynucleotide sequences encoding the75-amino acid GHRH protein described above driven by a 685 bp fragmentderived from the swine a-skeletal actin promoter (SEQ ID No. 3) wasconstructed as described below.

[0090] The oligonucleotide fragment encoding the 75 amino acid GHRHprotein (SEQ ID No. 1) was PCR-amplified from plasmid pGHRH4 using aprimer designated p97-S1 containing a Hind III site SEQ ID No. 4) and aprimer designated p97-A258, containing an Xba I site (SEQ ID No. 5). ThePCR-amplified sequence was then cloned into the Hind III-Xba I site ofplasmid pGL3 basic (Promega) to produce a plasmid designated GHRH1-44 SK(FIG. 2). A 685 bp fragment corresponding to a portion of the porcinea-skeletal actin promoter designated SK685 was PCR-amplified from swinegenomic DNA using primer designated SK-3, containing a Kpn I site (SEQID No. 6) and primer designated SK4, containing a Hind III site; (SEQ IDNo. 7). The PCR-amplified SK685 promoter fragment was then cloned intoplasmid GHRH1-44SK digested with Kpn I and HindIII enzymes to produce aplasmid designated GHRH1-44WTSK685 (FIG. 3).

[0091] 5. pGHRH1-44WTSK2014

[0092] A plasmid containing the polynucleotide sequences encoding the75-amino acid GHRH protein described above operatively linked to afragment derived from the swine a-skeletal actin promoter approximately2014 bp (SEQ ID No. 8) was constructed as described below.

[0093] An approximately 2014 bp fragment designated SK 2014corresponding to a portion of the porcine α-skeletal actin promoter wasPCR-amplified from swine genomic DNA using primer designated SK-7;containing a Kpn I site (SEQ ID No. 9) and primer designated SK-8;containing a Hind III site; (SEQ ID No. 10). The PCR-amplified SK2014promoter fragment was then cloned into plasmid GHRH1-44SK which wasdigested with Kpn I and Hind III enzymes to produce a plasmid designatedGHRH1-44WTSK2014 (FIG. 4).

[0094] 6. pGHRH1-29WTCMV

[0095] A plasmid containing the polynucleotide sequences encoding thesignal sequence derived from human GHRH polynucleotides and encodingamino acids 1-29 of swine GHRH protein was produced as described below.

[0096] An approximately 189 bp DNA fragment was PCR-amplified fromplasmid pGHRH-4 using primer designated GHRH-5, containing a Bam HIsite; (SEQ ID No. 11) and primer designated GHRH-6, containing a Bgl IIsite; (SEQ ID No. 12). The PCR-amplified fragment was digested with BamHI and Bgl II enzymes and cloned into plasmid pVR1012 (Vical, San Diego,Calif.) which was digested with Bam HI and Bgl II enzymes to produce aplasmid designated GHRH1-29WTCMV (SEQ ID No. 51) in which expression ofthe GHRH 1-29 protein is driven by CMV IE promoter/enhancer sequences.

[0097] 7. pGHRH1-29YWVTCMV

[0098] In order to produce novel variants of GHRH protein with enhancedstability to enzymatic degradation (e.g. DPPIV enzyme degradation) aplasmid containing polynucleotide sequences encoding the signal sequenceof human GHRH and an altered version of the 1-29 porcine GHRH proteinwas produced. The alteration consisted of the addition of an extratyrosine residue just preceding the first tyrosine residue of thenatural porcine GHRH 1-29 sequence. This modification alters the aminoterminal in such away that it is no longer recognized or cleaved byDPPIV enzyme. A plasmid containing the gene for this variant GHRHprotein was produced as described below.

[0099] A set of overlapping oligonucleotides (SEQ ID Nos. 13-25) weresynthesized, mixed, and GHRH was amplified using the PCR method. The PCRreaction resulted in the formation of a fragment of approximately 192 bpencoding amino acids 1-29 of GHRH in which nucleotides encoding atyrosine residue were inserted immediately 5′ to the coding sequence ofGHR(1-29) and further containing a Bam HI site (5′ end) and an Bgl IIsite (3′ end ). The 192 bp fragment was then digested with Bam HI andBgl II enzymes and cloned into plasmid pVR1012 which was digested withBam HI and Bgl II enzymes to produce plasmid designated GHRH1-29YWTCMV(SEQ ID No. 52) in which expression of GHRH1-29 (now 30) is driven byCMV IE promoter enhancer elements.

[0100] 8. pGHRH1-29YVTSK685

[0101] A plasmid containing the 192 bp fragment described above inSection 5 under the control of the SK685 promoter fragment was producedas described below.

[0102] The 192 bp fragment was amplified with two primers designatedp99-S1, containing a 5′ end Hind III site; (SEQ ID No. 26) and p99-A214,containing a 3′ end Xba I site; (SEQ ID No. 27). The PCR-amplifiedfragment was then digested with Hind III and Xba I enzymes and clonedinto plasmid GHRH1-29 Yala1522SK685 (see below) also digested with HindIII and Xba I enzymes to produce plasmid GHRH1-29YWTSK685 (SEQ ID No.53).

[0103] 9. pGHRH1-29YWTSK2014

[0104] A plasmid containing the 192 bp fragment described above inSection 5 under the control of the SK2014 promoter fragment was producedas described below.

[0105] The 192 bp fragment was amplified with two primers designatedp99-Si containing a 5′ end Hind III site; (SEQ ID No. 28) and p99-A214containing a 3′ end Xba I site; (SEQ ID No. 29). The PCR-amplifiedfragment was then digested with Hind III and Xba I enzymes and clonedinto plasmid GHRH1-29 YAla 1522SK2014 (see below) also digested withHind III and Xba I enzymes to produce plasmid GHRH1-29YWTSK2014 (SEQ IDNo. 54).

[0106] 10. pGHRH1-29YAla1522CMV

[0107] In order to produce novel variants of GHRH protein with enhancedstability to enzymatic degradation (e.g., due to DPPIV enzyme) andenhanced potency, a plasmid containing the signal sequence of human GHRHand an altered version of the 1-29 porcine GHRH protein was produced.The alteration consisted of the addition of an extra tyrosine residuejust preceding the first tyrosine residue of the natural porcine GHRH1-29 sequence and replacement of glycine 15 and leucine 22 with alanine.These modifications alter the amino terminal end of GHRH1-29 in suchaway that it is no longer by recognized or cleaved by the DPP IV enzymeand the modified protein has enhanced potency relative to the 29 or the44 amino acid GHRH protein. A plasmid containing the gene for thisvariant protein was produced as described below.

[0108] A set of overlapping oligonucleotides were synthesized (SEQ IDNos. 30-42), mixed and amplified using the PCR method. The PCR reactionresulted in the formation of a fragment of approximately 192 bpcontaining a Bam Hi site (5′ end) and an Bgl II site (3′ end) and inwhich an extra three nucleotides encoding for tyrosine is immediately 5′to the nucleotide sequence encoding the natural tyrosine at position 1of the GHRH1-29 sequence. Furthermore, the alteration includedreplacement of the 3 nucleotides encoding glycine 15 with threenucleotides encoding alanine and replacement of three nucleotidesencoding leucine 22 with three nucleotides encoding alanine. The 192 bpfragment was then digested with Bam HI and Bgl II enzymes and clonedinto plasmid pVR1012 which was digested with Bam HI and Bgl II enzymesto produce plasmid designated GHRH1-29YAla 1522CMV in which expressionof the variant GHRH1-29 (now 30) is driven by CMV IE promoter enhancerelements (SEQ ID No. 55).

[0109] 11. pGHRH1-29YAla15225K685

[0110] A plasmid containing the GHRH1-29YAla1522 fragment describedabove under the control of the SK685 promoter fragment was produced asdescribed below.

[0111] An approximately 192 bp fragment as described above in Section 8was PCR-amplified with primers designated p99-S1, containing a Hind IIIat 5′ end (SEQ ID No. 26) and p99-A214 containing a Xba I site at 3′end; (SEQ ID No. 27). The PCR-amplified fragment was digested with HindIII and Xba I enzymes and cloned into plasmid pGL3 (Promega) alsodigested with Hind III and Xba I to produce plasmid GHRH1-29YAla 1522SK(SEQ ID No. 56). A 685 bp fragment corresponding to a portion of theporcine a-skeletal actin promoter was PCR-amplified from swine genomicDNA using primers SK-3 (Kpn I site) and SK-4 (Hind III site). Thisfragment was designated SK685. The PCR-amplified SK685 promoter fragmentwas then cloned into plasmid GHRH1-29YAla1522SK digested with KpnI andHindIII enzymes to produce a plasmid designated GHRH1-29YA1a1522SK685(SEQ ID No. 57).

[0112] 12. pGHRH1-29Yala1522SK2014

[0113] A plasmid containing the GHRH1-29YAla1522 fragment describedabove under the control of the SK2014 promoter fragment was produced asdescribed below.

[0114] An approximately 192 bp fragment as described above in Section 8was PCR amplified with primers designated p99-S1 containing a Hind IIIat 5′ end; (SEQ ID No. 26) and p99-A214 containing a Xba I site at 3′end; (SEQ ID No. 27). The PCR-amplified fragment was digested with HindIII and Xba I enzymes and cloned into plasmid pGL3 (Promega) alsodigested with HindIII and Xba I to produce plasmid GHRH1-29YAla1522SK.An 2014 bp fragment corresponding to a portion of the porcine a-skeletalactin promoter was PCR-amplified from swine genomic DNA using primersSK-7 containing a Kpn I site and SK-8 containing a Hind III site. Thisfragment was designated SK2014. The PCR-amplified SK2014 promoterfragment was then cloned into plasmid GHRH1-29YAla1522SK digested withKpn I and Hind III enzymes to produce a plasmid designatedGHRH1-29YAla1522SK2014 (SEQ ID No. 58).

[0115] 13. pGHRH 1-44YWTCMV

[0116] In order to produce novel variants of GHRH protein with enhancedstability to enzymatic degradation (e.g., due to DPPIV enzyme) a plasmidcontaining the signal sequence of human GHRH and an altered version ofthe 1-44 porcine GHRH protein was produced. The alteration consisting ofthe addition of an extra tyrosine residue immediately 5′ to thenucleotide sequence encoding the first tyrosine residue of the naturalporcine GHRH 1-44 sequence. This modification will alter the aminoterminal end in such away that it is no longer by recognized or cleavedby DPPIV enzyme. A plasmid containing the gene for this modified GHRHprotein is produced as described below.

[0117] A set primers designated GHRH-1 (SEQ ID No. 61) and GHRH-3 (SEQID No. 65) were used in a PCR reaction amplify the human GHRH signalsequence and porcine GHRH from pGHRH1-29YWTCMV. The resulting GHRHfragment was digested with Bam HI and Bgl II, and cloned into the Bam HIsite of plasmid pVR1012 (Vical, San Diego, Calif.) to produce theplasmid designated GHRH1-44YWTCMV (SEQ ID No. 59).

[0118] 14. Synthesis of GH Constructs

[0119] In order to provide plasmid constructs containing GH genessuitable for treatment of growth hormone maladies or to enhance animalhealth and productivity, the construction of plasmids of the inventionand their methods of use are described below in detail.

[0120] The canine GH gene was cloned into plasmids vectors suitable forthe various aspects of the present invention using the followingprocedures. Total RNA was prepared from the pituitary gland of a dogusing the RNAzol B method using reagents and procedures from BiotecxLaboratories, Houston Tex. Briefly, about 0.15 mg of the tissue washomogenized in 2 ml of RNAzol B solution in a RNase-free glasshomogenizer. The material was then divided into two equal halves, andRNA extracted by chloroform and ethanol precipitation. The nucleic acidpellet was dried and taken up in RNase-free water. Reverse transcription(RT) of total RNA was done in a 20 ml volume using 0.02 mg of RNA, 138pmol of oligo #2 (SEQ ID No. 43), 1 mM MnCl₂, and the recommendedamounts of dNTPs and rTth enzyme from the RT-PCR kit purchased fromPerkin-Elmer, Norwalk, Conn. The reaction was incubated at 70□ for 11min in a Perkin-Elmer Thermal cycler. The completed (RT) reaction wasthen subjected to PCR following addition of 66 pmol of oligo #1 (SEQ IDNo. 44), 2.5 mM MgCl₂, and chelating buffer from the RT-PCR reactionkit. The PCR conditions were as follows: 94 C, 1 min; 55 C, 1 min; and72 C, 2 min; for 32 cycles. The □0.7 kb PCR-amplified DNA fragmentobtained was cloned into plasmid pCRScript purchased from Stratagene, LaJolla, Calif. and used according to manufacturer's recommendations. Therecombinant plasmid thus generated was termed cCG-SP. The insertfragment was partially sequenced and confirmed to contain the growthhormone (“GH”) DNA sequences. cGH-SP plasmid DNA was then used as atemplate to PCR-amplify using oligonucleotides oligo #3 (SEQ ID No. 45)and oligo #4 (SEQ ID No. 46) using reagents from the PCR system kit fromPerkin-Elmer using standard procedures and following cycling conditions:94 C, 1 min 1 cycle; 94 C, 30 sec; 55 C, 30 sec; 72 C, 1 min; 30 cycles.The □0.7 kb PCR-amplified DNA fragment obtained was subjected to columnpurification (Qiagen, Chatsworth, Calif.), and digested with restrictionenzymes EcoRV and BgIII by standard protocols (Sambrook et al., 1989).The digested PCR fragment was ligated to EcoRV-BgIII digested pCMV-MCS,a plasmid derivative of pCMVb (Clontech, Palo Alto, Calif.), engineeredto contain multiple cloning sites in place of lacZ gene. The ligationproduct was used to transform E. coli, and transformants were selectedfor resistance to ampicillin (pCMV-MCS-enooded marker). Transformantswere analyzed by plasmid DNA preparation and restriction site analysis,and a clone of the GH DNA sequences in pCMV-MCS was isolated (termedpCGH#9). The insert sequences were completely sequenced by standardprocedures to confirm presence of GH DNA sequences. The EcoRV-BgIII GHfragment from pCGH#9 has also been sub-cloned into gene therapy plasmidVR1012 (obtained from Vical, San Diego, Calf.). This clone referred toas pC51.

[0121] 15. Synthesis of GH-GHRH Constructs

[0122] A GH-GHRH fusion protein, comprising the carboxy terminal 20amino acids of GH and full length wildtype GHRH is produced. The fulllength GHRH gene is PCR amplified from plasmid pGHRH-4 using two primersdesignated GHRH-1 containing a Bgl II site (SEQ ID No. 61) and GHRH-2containing a Bam HI site (SEQ ID No. 62). The PCR-amplified fragment isthen cloned into the Bam HI site of the pVR1012 plasmid. Twocomplementary oligonucleotides encoding the carboxy terminal amino acids172-191 of GH (GH-1 oligo.; SEQ ID No. 62 and GH-2 oligo.; SEQ ID No.63) are synthesized. The GH-1 and GH-2 oligonucleotides are annealed andcloned into the Bam HI site of the pVR1012 plasmid containing the fulllength GHRH gene to produce pGHRH1-44WTGHpep (SEQ ID No. 60).

[0123] 16. In Vitro Studies Assessing GHRH Expression Levels

[0124] In order to assess the expression level of GHRH from pGHRH-4,this plasmid or. pVR1012 was transfected into C2C12 mouse myoblastsusing the fugene reagent according to the manufacturer=s recommendations(Boehringer Ingelheim). Supernatant harvested from transfected andnon-transfected cells at various time points were assayed for thepresence of GHRH using a commerically available radioimmune assay kit(Pennisula Laboratories). The results depicted in FIG. 5 indicate thatGHRH production could be detected 24 hours post-transfection.

[0125] 17. The Effect of GH Plasmid Injection on Swine Growth

[0126] In order to evaluate the effect of a single injection of plasmidscontaining GH gene on swine growth, experiments addressing the effect GHtreatment on swine of different ages were carried out according to theexperimental design described below.

[0127] Materials & Methods

[0128] Thirty six 3-week old (weaned) cross-bred (Yorkshire X landrace)pigs of mixed sex were brought into experimental barns and maintainedfor an acclimation period of 3 weeks. Animals were kept in pens (2/pen)according to treatment group. Animals were fed daily a non-medicatedcommercial pig diet ad libitum (16% protein pellet until approximately45 Kg body weight and then a 14% protein pellet until slaughter) andfresh water was available ad libitum. Animals were randomized into oneof four treatment groups (A-D; table 1) according to weight, sex andlitter. Eight animals in group A were injected once with plasmidscontaining GH gene at 6 weeks of age. Controls (10 animals; group C)were injected with blank plasmid vector when they were also 6 weeks ofage. Eight animals in group B were injected once with plasmidscontaining GH gene at 13 weeks of age. Controls (group D; 10 animals)were injected with blank plasmid vector when they were also 13 weeks ofage. Food consumption was recorded daily for each pen and animals wereweighed on two consecutive days each week until slaughter. Loin eye areaand back fat were quantified at slaughter. TABLE 1 Experimental DesignWeight at Number of Age at time of time of Group Animals Plasmid doseinjection injection A 8 4.6 mg  6 weeks 12.4 B 8 4.6 mg 13 weeks 4.2 C10 Placebo  6 weeks 13.8 D 10 Placebo 13 weeks 41.9

[0129] Results

[0130] As shown in table 2, treatment of 6 week-old pigs with plasmidsencoding GH gene results in enhanced performance. This is evident by the5% increase in weight gain and 5.1% increase in Average daily gain (ADG)achieved in GH injected animals versus placebo injected pigs. Moreover,the data shows that GH plasmid injection resulted in an improvement of3.3 % in feed efficiency (ratio of feed consumed relative to weightgain) as well as an increase of 7.2% in loin eye area of GH plasmidinjected animals.

[0131] The data in table 3 shows that treatment of 13 week-old pigs withplasmids encoding GH gene results in an even higher magnitude ofincrease in animal performance relative to age placebo injected controlsthan treatment of 6 week-old pigs. This is evident by the 9.5% increasein weight gain and 9.3% increase in Average daily gain (ADG) achieved inGH injected animals versus placebo injected pigs. Moreover, the datashows that GH plasmid injection resulted in an improvement of 6.7 % infeed efficiency (ratio of feed consumed relative to weight gain) as wellas an increase of 6.8% in loin eye area of GH plasmid injected animals.Thus treatment of animals with plasmids containing GH gene results inenhanced animal growth and performance. TABLE 2 The effect of GH plasmidadministration of weight gain Group A C % Improvement Weight atinjection 12.4 13.8 time (Kg) Weight gain^(a) 77.7 73.8 5.0% ADG^(b) .79.75 5.1% total feed 473.3 457.6 3.4% intake/pen^(c) Feed 4.8 4.7 2.1%intake/pen/day^(d) feed to gain^(e) 3.03 3.13 3.3% Loin eye area at39.51 36.83 7.2% Slaughter (Cm²)

[0132] TABLE 3 The effect of GH plasmid administration of weight gainGroup B D % Improvement Weight at injection 42.0 41.9 N/A time (Kg)Weight gain^(a) 52.1 47.6 9.5% ADG^(b) 1.06 .97 9.3% total feed 309.6300.7 3.0% intake/pen^(c) Feed 6.3 6.1 3.3% intake/pen/day^(d) feed togain^(e) 3.0 3.2 6.7% Loin eye area at 41.61 38.96 6.8% Slaughter (Cm²)

[0133] A number of references have been cited and the entire disclosuresof which are incorporated herein by reference.

[0134] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

1 67 1 246 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 1 ggatccgcca ccatgccact ctgggtgttc ttctttgtga tcctcaccctcagcaacagc 60 tcccactgct ccccacctcc ccctttgacc ctcaggatgc ggcggtatgcagatgccatc 120 ttcaccaaca gctaccggaa ggtgctgggc cagctgtccg cccgcaagctgctccaggac 180 atcatgagca ggcagcaggg agagagaaac caagagcaag gagcaagggtgcggctttga 240 agatct 246 2 75 PRT Artificial Sequence Description ofArtificial Sequence GHRH protein 2 Met Pro Leu Trp Val Phe Phe Phe ValIle Leu Thr Leu Ser Asn Ser 1 5 10 15 Ser His Cys Ser Pro Pro Pro ProLeu Thr Leu Arg Met Arg Arg Tyr 20 25 30 Ala Asp Ala Ile Phe Thr Asn SerTyr Arg Lys Val Leu Gly Gln Leu 35 40 45 Ser Ala Arg Lys Leu Leu Gln AspIle Met Ser Arg Gln Gln Gly Glu 50 55 60 Arg Asn Gln Glu Gln Gly Ala ArgVal Arg Leu 65 70 75 3 685 DNA Artificial Sequence Description ofArtificial Sequence obligonucleotide 3 ggtaccatcg ctggggagct gggggaggggtcgccttcct gccctaccca ggactccggg 60 tgcgaccgct cctctatctc tccagcccaccaccactcca ccacttggac acgtctccct 120 cctccctgga gtcgctctag agggtttgggggtctgagta aagaacccga agtagggata 180 cagtgtggcg gcaccttcca gaggccccgggcgcagggta gaccggggcg gggcggcccg 240 cggacaggtg cagccccagg cgcaggcgcactcgcgcctc ccggcgcagg cggtgaacct 300 cgccccaccc cagcccctcc ggggggcagctgggccgggt cgggaggggc ccaccagccc 360 gggagacact ccatatacgg ccaggcccgctttacctggg ctccggccag gccgctcctt 420 ctttggtcag cacaggggac ccgggcgggggcccaggccg ctaacccgcc gggggagggg 480 gctccagtgc ccaacaccca aatatggctcgagaagggga gcgacattcc agtgaggcgg 540 ctcgggggga gaacccgcgg gctatataaaacctgagcgt ggggaccagc ggccaccgca 600 gcggacagcg ccgagagaag cctcgcttccctcccgcggc gaccagggcc ccagccggag 660 agcagcaggt gtagccacca agctt 685 430 DNA Artificial Sequence Description of Artificial Sequence primer 4aatcccaagc ttgccaccat gccactctgg 30 5 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 5 tattgctcta gatcaaagccgcacccttgc 30 6 39 DNA Artificial Sequence Description of ArtificialSequence Primer 6 cgggtaccat cgctggggga gctggggcag gggtcgcct 39 7 40 DNAArtificial Sequence Description of Artificial Sequence Primer 7cccgcttggt ggctacacct gctgctctcc ggctggggcc 40 8 2014 DNA ArtificialSequence Description of Artificial Sequence SK 2014 8 ggtaccgctataggagagaa aagagctgca ctgagcaccc tccttcccct ttaaatgtca 60 acagattaggagtcagtgaa tgacagcaca cctcttgcta ccttagagac caaaatttaa 120 gctactccccttaagctata gctagagtgc acctgccagt gtctttagtc cccactgatg 180 gaacaggacccaaggtattg aagatggaac atagttattc attcatcctc taatttaaaa 240 agctggatatgctgtacagc agaaattgac ggaacaatgt aaatcaacta taacagaaga 300 aataaaaacctggggggaaa gaagctgact atgaaacccc aggagctttc tacatgggcc 360 tggactcaccaaactcttta ttttgtaatg gacttctgac atttttagga agggctgtcc 420 tgatgtgggctatagaagag ggtttcacat gcttcttcaa gaggacccac actgtcccag 480 ttgctgagtcccaccaccag atgctagtgg cagctatttg gggaacactt aggcactaca 540 aaaaaatgagtgattccatt ctggctcaca ccatatccct gatgtacccc ttaaagcatg 600 tcactgagttcatcacagaa aattgtttcc cctgtgcctt ccacaacaag gttagagctg 660 tccttggggccaggggaagg gggcagggag tgagaagcac caactggata acctcctctg 720 acccccactccaccttacca taagtagatc caaatccttc tagaaaatta ggaaggcata 780 tccccatatatcagcgatat aaatagaact gcttcagcgc tctggtagac ggtgactctc 840 caaggtggactgggaggcag cctggccttg gctgggcatc gtcctctaaa tagaaagatg 900 aacttgttcagcctttccag aaggaaaact gctgcccagc ctacagtgca acgtccttgt 960 cttccatctggaggaagcac gggtgacata tcatctagta agggcacctc tctgtttcca 1020 cctccaggtcgaggggtgtg acccttactt ctcagcctca agggagggac actcaacycc 1080 ccaaaaagacatgagggcgc tcagctcggc ccaccgcacc ccggaccgga gccgtcaccc 1140 cccgaaattcactcccttca caagccccca agcgcgttct ctggtgcgga ctgctccggg 1200 gccctggctttgtgcccagc gttgtcagag ccaccgccct gagcctgtcc ccgggagccc 1260 cgcgcctcctcccaccgctc crctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320 ctgcgcgccctcccgctgcc ggtggccctc tccggtgggg gtggggaccg acagggtcag 1380 ccctccggatccggggcgct ccgggtagcg gggagaagtg atcgctgggg agctggggga 1440 ggggtcgccttcctgcccta cccaggactc cgggtgcgac cgctcctcta tctctccagc 1500 ccaccaccactccaccactt ggacacgtct ccctcctccc tggagtcgct ctagagggtt 1560 tgggggtctgagtaaagaac ccgaagtagg gatacagtgt ggcggcacct tccagaggcc 1620 ccgggcgcagggtagaccgg ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1680 cgcactcgcgcctcccggcg caggcggtga acctcgcccc accccagccc ctccgggggg 1740 cagctgggccgggtcgggag gggcccacca gcccgggaga cactccatat acggccaggc 1800 ccgctttacctgggctccgg ccaggccgct ccttctttgg tcagcacagg ggacccgggc 1860 gggggcccaggccgctaacc cgccggggga gggggctcca gtgcccaaca cccaaatatg 1920 gctcgagaaggggagcgaca ttccagtgag gcggctcggg gggagaaccc gcgggctata 1980 taaaacctgagcgtggggac cagcggccaa gctt 2014 9 30 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 9 cccaagcttg gccgctggtc cccacgctca 30 1038 DNA Artificial Sequence Description of Artificial Sequence Primer 10caggtaccgc tataggagac aaaagagtgc actgagca 38 11 46 DNA ArtificialSequence Description of Artificial Sequence Primer 11 agatatcccggccgctctag accaggcccc tggatccgcc accatg 46 12 44 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 gaagatctct acctgctcatgatgtcctgg agcagcttgc gggc 44 13 20 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 13 gtcattccga gattcggata 20 14 40 DNAArtificial Sequence Description of Artificial Sequence Primer 14gtcattccga gattcggata cacaggatcc gccaccatcc 40 15 40 DNA ArtificialSequence Description of Artificial Sequence Primer 15 cactctgggtgttcttcttt gtgatcctca ccctcagcaa 40 16 40 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 cagctcccac tgctccccacctcccccttt gaccctcagg 40 17 40 DNA Artificial Sequence Description ofArtificial Sequence Primer 17 atgcggcggt attatgcaga tgccatcttcaccaacagct 40 18 40 DNA Artificial Sequence Description of ArtificialSequence Primer 18 accggaaggt gctgggccag ctgtccgccc gcaagctcct 40 19 40DNA Artificial Sequence Description of Artificial Sequence Primer 19ccaggacatc atgagcaggt agagatctga taagcgttat 40 20 20 DNA ArtificialSequence Description of Artificial Sequence Primer 20 ataacgcttatcagatctct 20 21 40 DNA Artificial Sequence Description of ArtificialSequence Primer 21 acctgctcat gatgtcctgg accagcttgc gggcggacag 40 22 40DNA Artificial Sequence Description of Artificial Sequence Primer 22ctggcccagc accttccggt agctgttggt gaagatggca 40 23 40 DNA ArtificialSequence Description of Artificial Sequence Primer 23 tctgcataataccgccgcat cctgagggtc aaacccccag 40 24 40 DNA Artificial SequenceDescription of Artificial Sequence Primer 24 gtggggagca gtgggagctgttgctgaggg tgaggatcac 40 25 40 DNA Artificial Sequence Description ofArtificial Sequence Primer 25 gtggggagca gtgggagctg ttgctgagggtcaggatcac 40 26 30 DNA Artificial Sequence Description of ArtificialSequence Primer 26 gagctcaagc ttgccaccat gccactctgg 30 27 28 DNAArtificial Sequence Description of Artificial Sequence Primer 27aagatctaga ctacctgctc atgatgtc 28 28 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 28 gagctcaagc ttgccaccatgccactctgg 30 29 28 DNA Artificial Sequence Description of ArtificialSequence Primer 29 aagatctaga ctacctgctc atgatgtc 28 30 20 DNAArtificial Sequence Description of Artificial Sequence Primer 30gtcattccga gattcggata 20 31 40 DNA Artificial Sequence Description ofArtificial Sequence Primer 31 gtcattccga gattcggata cacaggatccgccaccatcc 40 32 40 DNA Artificial Sequence Description of ArtificialSequence Primer 32 cactctgggt gttcttcttt gtgatcctca ccctcagcaa 40 33 39DNA Artificial Sequence Description of Artificial Sequence Primer 33cagctccact gctccccacc tccccctttg accctcagg 39 34 40 DNA ArtificialSequence Description of Artificial Sequence Primer 34 atgcggcggtattatgcaga tcccatcttc accaacagct 40 35 40 DNA Artificial SequenceDescription of Artificial Sequence Primer 35 accggaaggt gctggcccagctgtccgccc gcaaggccct 40 36 40 DNA Artificial Sequence Description ofArtificial Sequence Primer 36 ccaggacatc atgagcaggt agagatctgataagcgttat 40 37 20 DNA Artificial Sequence Description of ArtificialSequence Primer 37 ataacgctta tcagatctct 20 38 40 DNA ArtificialSequence Description of Artificial Sequence Primer 38 acctgctcatgatgtcctgg agggccttgc gggcccacag 40 39 40 DNA Artificial SequenceDescription of Artificial Sequence Primer 39 ctgggccagc accttccggtacctgttggt gaagatggca 40 40 40 DNA Artificial Sequence Description ofArtificial Sequence Primer 40 tctccataat accgccgcat cctgagggtcaaagggggag 40 41 40 DNA Artificial Sequence Description of ArtificialSequence Primer 41 gtggggagca gtgggagctg ttgctgaggg tgaggatcac 40 42 40DNA Artificial Sequence Description of Artificial Sequence Primer 42gtggggagca gtgggagctc ttgctgaggg tgaggatcac 40 43 25 DNA ArtificialSequence Description of Artificial Sequence Primer 43 ctagaaggcacagctgcttt ccacg 25 44 25 DNA Artificial Sequence Description ofArtificial Sequence Primer 44 atggctgcag gcccccggac ctctg 25 45 27 DNAArtificial Sequence Description of Artificial Sequence Primer 45aaagatatca tggctgcagg cccccgg 27 46 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 46 aaaagatctc tagaaggcacagctgct 27 47 5185 DNA Artificial Sequence Description of ArtificialSequence pGHRH-4 construct 47 gctgtgcctt ctagttgcca gccatctgttgtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcctaataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggtggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggatgcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc ctgggccagaaagaagcagg cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagttccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgctaaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac ctagcctccaagagtgggaa gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctccaacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactgactcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaatacggttatcc acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagcaaaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccctgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga caggactataaagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgccgcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctcacgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacgaaccccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaacccggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgaggtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct acactagaagaacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtagctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagcagattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta cggggtctgacgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggatcttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatgagtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct cagcgatctgtctatttcgt tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcgtgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagtgagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga ttttgaacttttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactcagcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgccagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat caaatgaaactgcaatttat tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaatgaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcgattccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa aataaggttatcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgcatttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgcatcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctgttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgcatcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccggggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtcggaagaggca taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattggcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaatcgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaatcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatggctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgatatatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg gctttccccccccccccatt attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaatgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacctgacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg tatcacgaggccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag agcagattgtactgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccgcatcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttatattggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatgacgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg ggtggagtatttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccctattgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgggactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaatgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgcttccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattattgaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg gctctttgccacaactatct ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactctgtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccgtcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga atctcgggtacgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggtcccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca gtggaggccagacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtagggtatgtgtc tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagacttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcagaggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcgttgctgccgc gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttccatgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg gccgctctagaccaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccctcagcaacag ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtatgcagatgccat cttcaccaac agctaccgga 5040 aggtgctggg ccagctgtcc gcccgcaagctgctccagga catcatgagc aggcagcagg 5100 gagagagaaa ccaagagcaa ggagcaagggtgcggctttg aagatcttag tagtagtagg 5160 cggccgctct agaggatcca gatct 518548 3369 DNA Artificial Sequence Description of Artificial SequencepGHRH1-44SK construct 48 ggtaccgagc tcttacgcgt gctagcccgg gctcgagatctgcgatctaa gtaagcttgc 60 caccatgcca ctctgggtgt tcttctttgt gatcctcaccctcagcaaca gctcccactg 120 ctccccacct ccccctttga ccctcaggat gcggcggtatgcagatgcca tcttcaccaa 180 cagctaccgg aaggtgctgg gccagctgtc cgcccgcaagctgctccagg acatcatgag 240 caggcagcag ggagagagaa accaagagca aggagcaagggtgcggcttt gatctagagt 300 cggggcggcc ggccgcttcg agcagacatg ataagatacattgatgagtt tggacaaacc 360 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaatttgtgatgc tattgcttta 420 tttgtaacca ttataagctg caataaacaa gttaacaacaacaattgcat tcattttatg 480 tttcaggttc agggggaggt gtgggaggtt ttttaaagcaagtaaaacct ctacaaatgt 540 ggtaaaatcg ataaggatcc gtcgaccgat gcccttgagagccttcaacc cagtcagctc 600 cttccggtgg gcgcggggca tgactatcgt cgccgcacttatgactgtct tctttatcat 660 gcaactcgta ggacaggtgc cggcagcgct cttccgcttcctcgctcact gactcgctgc 720 gctcggtcgt tcggctgcgg cgagcggtat cagctcactcaaaggcggta atacggttat 780 ccacagaatc aggggataac gcaggaaaga acatgtgagcaaaaggccag caaaaggcca 840 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccataggctccgcccc cctgacgagc 900 atcacaaaaa tcgacgctca agtcagaggt ggcgaaacccgacaggacta taaagatacc 960 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgttccgaccctg ccgcttaccg 1020 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgctttctcaatgc tcacgctgta 1080 ggtatctcag ttcggtgtag gtcgttcgct ccaagctgggctgtgtgcac gaaccccccg 1140 ttcagcccga ccgctgcgcc ttatccggta actatcgtcttgagtccaac ccggtaagac 1200 acgacttatc gccactggca gcagccactg gtaacaggattagcagagcg aggtatgtag 1260 gcggtgctac agagttcttg aagtggtggc ctaactacggctacactaga aggacagtat 1320 ttggtatctg cgctctgctg aagccagtta ccttcggaaaaagagttggt agctcttgat 1380 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgtttgcaagcag cagattacgc 1440 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttctacggggtct gacgctcagt 1500 ggaacgaaaa ctcacgttaa gggattttgg tcatgagattatcaaaaagg atcttcacct 1560 agatcctttt aaattaaaaa tgaagtttta aatcaatctaaagtatatat gagtaaactt 1620 ggtctgacag ttaccaatgc ttaatcagtg aggcacctatctcagcgatc tgtctatttc 1680 gttcatccat agttgcctga ctccccgtcg tgtagataactacgatacgg gagggcttac 1740 catctggccc cagtgctgca atgataccgc gagacccacgctcaccggct ccagatttat 1800 cagcaataaa ccagccagcc ggaagggccg agcgcagaagtggtcctgca actttatccg 1860 cctccatcca gtctattaat tgttgccggg aagctagagtaagtagttcg ccagttaata 1920 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggtgtcacgctcg tcgtttggta 1980 tggcttcatt cagctccggt tcccaacgat caaggcgagttacatgatcc cccatgttgt 2040 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgtcagaagtaag ttggccgcag 2100 tgttatcact catggttatg gcagcactgc ataattctcttactgtcatg ccatccgtaa 2160 gatgcttttc tgtgactggt gagtactcaa ccaagtcattctgagaatag tgtatgcggc 2220 gaccgagttg ctcttgcccg gcgtcaatac gggataataccgcgccacat agcagaactt 2280 taaaagtgct catcattgga aaacgttctt cggggcgaaaactctcaagg atcttaccgc 2340 tgttgagatc cagttcgatg taacccactc gtgcacccaactgatcttca gcatctttta 2400 ctttcaccag cgtttctggg tgagcaaaaa caggaaggcaaaatgccgca aaaaagggaa 2460 taagggcgac acggaaatgt tgaatactca tactcttcctttttcaatat tattgaagca 2520 tttatcaggg ttattgtctc atgagcggat acatatttgaatgtatttag aaaaataaac 2580 aaataggggt tccgcgcaca tttccccgaa aagtgccacctgacgcgccc tgtagcggcg 2640 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgaccgctacactt gccagcgccc 2700 tagcgcccgc tcctttcgct ttcttccctt cctttctcgccacgttcgcc ggctttcccc 2760 gtcaagctct aaatcggggg ctccctttag ggttccgatttagtgcttta cggcacctcg 2820 accccaaaaa acttgattag ggtgatggtt cacgtagtgggccatcgccc tgatagacgg 2880 tttttcgccc tttgacgttg gagtccacgt tctttaatagtggactcttg ttccaaactg 2940 gaacaacact caaccctatc tcggtctatt cttttgatttataagggatt ttgccgattt 3000 cggcctattg gttaaaaaat gagctgattt aacaaaaatttaacgcgaat tttaacaaaa 3060 tattaacgtt tacaatttcc cattcgccat tcaggctgcgcaactgttgg gaagggcgat 3120 cggtgcgggc ctcttcgcta ttacgccagc ccaagctaccatgataagta agtaatatta 3180 aggtacggga ggtacttgga gcggccgcaa taaaatatctttattttcat tacatctgtg 3240 tgttggtttt ttgtgtgaat cgatagtact aacatacgctctccatcaaa acaaaacgaa 3300 acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagtgcaggtgcca gaacatttct 3360 ctatcgata 3369 49 3976 DNA ArtificialSequence Description of Artificial Sequence pGHRH1-44WTSK685 construct49 ggtaccatcg ctggggagct gggggagggg tcgccttcct gccctaccca ggactccggg 60tgcgaccgct cctctatctc tccagcccac caccactcca ccacttggac acgtctccct 120cctccctgga gtcgctctag agggtttggg ggtctgagta aagaacccga agtagggata 180cagtgtggcg gcaccttcca gaggccccgg gcgcagggta gaccggggcg gggcggcccg 240cggacaggtg cagccccagg cgcaggcgca ctcgcgcctc ccggcgcagg cggtgaacct 300cgccccaccc cagcccctcc ggggggcagc tgggccgggt cgggaggggc ccaccagccc 360gggagacact ccatatacgg ccaggcccgc tttacctggg ctccggccag gccgctcctt 420ctttggtcag cacaggggac ccgggcgggg gcccaggccg ctaacccgcc gggggagggg 480gctccagtgc ccaacaccca aatatggctc gagaagggga gcgacattcc agtgaggcgg 540ctcgggggga gaacccgcgg gctatataaa acctgagcgt ggggaccagc ggccaccgca 600gcggacagcg ccgagagaag cctcgcttcc ctcccgcggc gaccagggcc ccagccggag 660agcagcaggt gtagccacca agcttgccac catgccactc tgggtgttct tctttgtgat 720cctcaccctc agcaacagct cccactgctc cccacctccc cctttgaccc tcaggatgcg 780gcggtatgca gatgccatct tcaccaacag ctaccggaag gtgctgggcc agctgtccgc 840ccgcaagctg ctccaggaca tcatgagcag gcagcaggga gagagaaacc aagagcaagg 900agcaagggtg cggctttgat ctagagtcgg ggcggccggc cgcttcgagc agacatgata 960agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 1020tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 1080aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt 1140taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatccgtc gaccgatgcc 1200cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc 1260cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt 1320ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 1380ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 1440tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 1500tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 1560gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1620ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 1680tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 1740agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 1800atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 1860acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1920actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 1980tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 2040tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 2100tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 2160tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 2220caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 2280cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 2340agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 2400acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 2460gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 2520ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 2580tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 2640ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 2700tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 2760attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 2820agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 2880ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 2940ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3000cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3060gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 3120tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 3180tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 3240tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 3300gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 3360ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 3420tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 3480gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 3540ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 3600ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 3660aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcccat tcgccattca 3720ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca 3780agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa 3840aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac 3900atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag 3960tgcaagtgca ggtgcc 3976 50 5325 DNA Artificial Sequence Description ofArtificial Sequence pGHRH1-44WTSK2014 construct 50 ggtaccgcta taggagagaaaagagctgca ctgagcaccc tccttcccct ttaaatgtca 60 acagattagg agtcagtgaatgacagcaca cctcttgcta ccttagagac caaaatttaa 120 gctactcccc ttaagctatagctagagtgc acctgccagt gtctttagtc cccactgatg 180 gaacaggacc caaggtattgaagatggaac atagttattc attcatcctc taatttaaaa 240 agctggatat gctgtacagcagaaattgac ggaacaatgt aaatcaacta taacagaaga 300 aataaaaacc tggggggaaagaagctgact atgaaacccc aggagctttc tacatgggcc 360 tggactcacc aaactctttattttgtaatg gacttctgac atttttagga agggctgtcc 420 tgatgtgggc tatagaagagggtttcacat gcttcttcaa gaggacccac actgtcccag 480 ttgctgagtc ccaccaccagatgctagtgg cagctatttg gggaacactt aggcactaca 540 aaaaaatgag tgattccattctggctcaca ccatatccct gatgtacccc ttaaagcatg 600 tcactgagtt catcacagaaaattgtttcc cctgtgcctt ccacaacaag gttagagctg 660 tccttggggc caggggaagggggcagggag tgagaagcac caactggata acctcctctg 720 acccccactc caccttaccataagtagatc caaatccttc tagaaaatta ggaaggcata 780 tccccatata tcagcgatataaatagaact gcttcagcgc tctggtagac ggtgactctc 840 caaggtggac tgggaggcagcctggccttg gctgggcatc gtcctctaaa tagaaagatg 900 aacttgttca gcctttccagaaggaaaact gctgcccagc ctacagtgca acgtccttgt 960 cttccatctg gaggaagcacgggtgacata tcatctagta agggcacctc tctgtttcca 1020 cctccaggtc gaggggtgtgacccttactt ctcagcctca agggagggac actcaacccc 1080 ccaaaaagac atgagggcgctcagctcggc ccaccgcacc ccggaccgga gccgtcaccc 1140 cccgaaattc actcccttcacaagccccca agcgcgttct ctggtgcgga ctgctccggg 1200 gccctggctt tgtgcccagcgttgtcagag ccaccgccct gagcctgtcc ccgggagccc 1260 cgcgcctcct cccaccgctccgctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320 ctgcgcgccc tcccgctgccggtggccctc tccggtgggg gtggggaccg acagggtcag 1380 ccctccggat ccggggcgctccgggtagcg gggagaagtg atcgctgggg agctggggga 1440 ggggtcgcct tcctgccctacccaggactc cgggtgcgac cgctcctcta tctctccagc 1500 ccaccaccac tccaccacttggacacgtct ccctcctccc tggagtcgct ctagagggtt 1560 tgggggtctg agtaaagaacccgaagtagg gatacagtgt ggcggcacct tccagaggcc 1620 ccgggcgcag ggtagaccggggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1680 cgcactcgcg cctcccggcgcaggcggtga acctcgcccc accccagccc ctccgggggg 1740 cagctgggcc gggtcgggaggggcccacca gcccgggaga cactccatat acggccaggc 1800 ccgctttacc tgggctccggccaggccgct ccttctttgg tcagcacagg ggacccgggc 1860 gggggcccag gccgctaacccgccggggga gggggctcca gtgcccaaca cccaaatatg 1920 gctcgagaag gggagcgacattccagtgag gcggctcggg gggagaaccc gcgggctata 1980 taaaacctga gcgtggggaccagcggccaa gcttgccacc atgccactct gggtgttctt 2040 ctttgtgatc ctcaccctcagcaacagctc ccactgctcc ccacctcccc ctttgaccct 2100 caggatgcgg cggtatgcagatgccatctt caccaacagc taccggaagg tgctgggcca 2160 gctgtccgcc cgcaagctgctccaggacat catgagcagg cagcagggag agagaaacca 2220 agagcaagga gcaagggtgcggctttgatc tagagtcggg gcggccggcc gcttcgagca 2280 gacatgataa gatacattgatgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 2340 tgctttattt gtgaaatttgtgatgctatt gctttatttg taaccattat aagctgcaat 2400 aaacaagtta acaacaacaattgcattcat tttatgtttc aggttcaggg ggaggtgtgg 2460 gaggtttttt aaagcaagtaaaacctctac aaatgtggta aaatcgataa ggatccgtcg 2520 accgatgccc ttgagagccttcaacccagt cagctccttc cggtgggcgc ggggcatgac 2580 tatcgtcgcc gcacttatgactgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 2640 agcgctcttc cgcttcctcgctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 2700 cggtatcagc tcactcaaaggcggtaatac ggttatccac agaatcaggg gataacgcag 2760 gaaagaacat gtgagcaaaaggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 2820 tggcgttttt ccataggctccgcccccctg acgagcatca caaaaatcga cgctcaagtc 2880 agaggtggcg aaacccgacaggactataaa gataccaggc gtttccccct ggaagctccc 2940 tcgtgcgctc tcctgttccgaccctgccgc ttaccggata cctgtccgcc tttctccctt 3000 cgggaagcgt ggcgctttctcaatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3060 ttcgctccaa gctgggctgtgtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3120 ccggtaacta tcgtcttgagtccaacccgg taagacacga cttatcgcca ctggcagcag 3180 ccactggtaa caggattagcagagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3240 ggtggcctaa ctacggctacactagaagga cagtatttgg tatctgcgct ctgctgaagc 3300 cagttacctt cggaaaaagagttggtagct cttgatccgg caaacaaacc accgctggta 3360 gcggtggttt ttttgtttgcaagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3420 atcctttgat cttttctacggggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3480 ttttggtcat gagattatcaaaaaggatct tcacctagat ccttttaaat taaaaatgaa 3540 gttttaaatc aatctaaagtatatatgagt aaacttggtc tgacagttac caatgcttaa 3600 tcagtgaggc acctatctcagcgatctgtc tatttcgttc atccatagtt gcctgactcc 3660 ccgtcgtgta gataactacgatacgggagg gcttaccatc tggccccagt gctgcaatga 3720 taccgcgaga cccacgctcaccggctccag atttatcagc aataaaccag ccagccggaa 3780 gggccgagcg cagaagtggtcctgcaactt tatccgcctc catccagtct attaattgtt 3840 gccgggaagc tagagtaagtagttcgccag ttaatagttt gcgcaacgtt gttgccattg 3900 ctacaggcat cgtggtgtcacgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3960 aacgatcaag gcgagttacatgatccccca tgttgtgcaa aaaagcggtt agctccttcg 4020 gtcctccgat cgttgtcagaagtaagttgg ccgcagtgtt atcactcatg gttatggcag 4080 cactgcataa ttctcttactgtcatgccat ccgtaagatg cttttctgtg actggtgagt 4140 actcaaccaa gtcattctgagaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4200 caatacggga taataccgcgccacatagca gaactttaaa agtgctcatc attggaaaac 4260 gttcttcggg gcgaaaactctcaaggatct taccgctgtt gagatccagt tcgatgtaac 4320 ccactcgtgc acccaactgatcttcagcat cttttacttt caccagcgtt tctgggtgag 4380 caaaaacagg aaggcaaaatgccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4440 tactcatact cttcctttttcaatattatt gaagcattta tcagggttat tgtctcatga 4500 gcggatacat atttgaatgtatttagaaaa ataaacaaat aggggttccg cgcacatttc 4560 cccgaaaagt gccacctgacgcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 4620 ttacgcgcag cgtgaccgctacacttgcca gcgccctagc gcccgctcct ttcgctttct 4680 tcccttcctt tctcgccacgttcgccggct ttccccgtca agctctaaat cgggggctcc 4740 ctttagggtt ccgatttagtgctttacggc acctcgaccc caaaaaactt gattagggtg 4800 atggttcacg tagtgggccatcgccctgat agacggtttt tcgccctttg acgttggagt 4860 ccacgttctt taatagtggactcttgttcc aaactggaac aacactcaac cctatctcgg 4920 tctattcttt tgatttataagggattttgc cgatttcggc ctattggtta aaaaatgagc 4980 tgatttaaca aaaatttaacgcgaatttta acaaaatatt aacgtttaca atttcccatt 5040 cgccattcag gctgcgcaactgttgggaag ggcgatcggt gcgggcctct tcgctattac 5100 gccagcccaa gctaccatgataagtaagta atattaaggt acgggaggta cttggagcgg 5160 ccgcaataaa atatctttattttcattaca tctgtgtgtt ggttttttgt gtgaatcgat 5220 agtactaaca tacgctctccatcaaaacaa aacgaaacaa aacaaactag caaaataggc 5280 tgtccccagt gcaagtgcaggtgccagaac atttctctat cgata 5325 51 5108 DNA Artificial SequenceDescription of Artificial Sequence pGHRH1-29WTCMV construct 51gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 60ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 180tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac ccaggtgctg 240aagaattgac ccggttcctc ctgggccaga aagaagcagg cacatcccct tctctgtgac 300acaccctgtc cacgcccctg gttcttagtt ccagccccac tcataggaca ctcatagctc 360aggagggctc cgccttcaat cccacccgct aaagtacttg gagcggtctc tccctccctc 420atcagcccac caaaccaaac ctagcctcca agagtgggaa gaaattaaag caagataggc 480tattaagtgc agagggagag aaaatgcctc caacatgtga ggaagtaatg agagaaatca 540tagaatttct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 600agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 660aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 720gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 780tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 840cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 900ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 960cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1020atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1080agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1140gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 1200gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1260tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1320agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1380gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 1440aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 1500aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 1560cggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 1620ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt 1680gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc 1740gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg 1800ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat 1860tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 1920ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 1980aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct 2040attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact 2100gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag 2160ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc 2220gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 2280tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 2340tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca 2400tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt 2460agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac 2520aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca 2580ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc 2640ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 2700taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag 2760agattttgag acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt 2820tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 2880ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 2940ttaacctata aaaataggcg tatcacgagg ccctttcgtc ctcgcgcgtt tcggtgatga 3000cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 3060tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg 3120gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 3180accgcacaga tgcgtaagga gaaaataccg catcagattg gctattggcc attgcatacg 3240ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt 3300tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 3360ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 3420aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 3480actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 3540caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 3600tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 3660ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3720cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3780tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3840atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3900cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 3960tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 4020gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 4080tttttggctt ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta 4140gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc 4200cattactaat ccataacatg gctctttgcc acaactatct ctattggcta tatgccaata 4260ctctgtcctt cagagactga cacggactct gtatttttac aggatggggt cccatttatt 4320atttacaaat tcacatatac aacaacgccg tcccccgtgc ccgcagtttt tattaaacat 4380agcgtgggat ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta 4440gcggcggagc ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg 4500gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca 4560ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag cgtggagatt 4620gggctcgcac ggctgacgca gatggaagac ttaaggcagc ggcagaagaa gatgcaggca 4680gctgagttgt tgtattctga taagagtcag aggtaactcc cgttgcggtg ctgttaacgg 4740tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac 4860acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc accatgccac 4920tctgggtgtt cttctttgtg atcctcaccc tcagcaacag ctcccactgc tccccacctc 4980cccctttgac cctcaggatg cggcggtatg cagatgccat cttcaccaac agctaccgga 5040aggtgctggg ccagctgtcc gcccgcaagc tgctccagga catcatgagc aggtagagat 5100ccagatct 5108 52 5108 DNA Artificial Sequence Description of ArtificialSequence pGHRH1-29YWTCMV construct 52 gctgtgcctt ctagttgcca gccatctgttgtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcctaataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggtggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggatgcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc ctgggccagaaagaagcagg cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagttccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgctaaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac ctagcctccaagagtgggaa gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctccaacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactgactcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaatacggttatcc acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagcaaaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccctgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga caggactataaagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgccgcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctcacgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacgaaccccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaacccggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgaggtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct acactagaagaacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtagctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagcagattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta cggggtctgacgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggatcttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatgagtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct cagcgatctgtctatttcgt tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcgtgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagtgagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga ttttgaacttttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactcagcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgccagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat caaatgaaactgcaatttat tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaatgaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcgattccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa aataaggttatcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgcatttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgcatcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctgttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgcatcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccggggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtcggaagaggca taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattggcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaatcgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaatcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatggctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgatatatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg gctttccccccccccccatt attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaatgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacctgacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg tatcacgaggccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag agcagattgtactgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccgcatcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttatattggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatgacgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg ggtggagtatttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccctattgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgggactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaatgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgcttccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattattgaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg gctctttgccacaactatct ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactctgtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccgtcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga atctcgggtacgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggtcccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca gtggaggccagacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtagggtatgtgtc tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagacttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcagaggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcgttgctgccgc gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttccatgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg gccgctctagaccaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccctcagcaacag ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtatgcagatgccat cttcaccaac agctaccgga 5040 aggtgctggg ccagctgtcc gcccgcaagctcctccagga catcatgagc aggtagagat 5100 ccagatct 5108 53 3954 DNAArtificial Sequence Description of Artificial Sequence pGHRH1-29YWTSK685construct 53 ggtaccatcg ctggggagct gggggagggg tcgccttcct gccctacccaggactccggg 60 tgcgaccgct cctctatctc tccagcccac caccactcca ccacttggacacgtctccct 120 cctccctgga gtcgctctag agggtttggg ggtctgagta aagaacccgaagtagggata 180 cagtgtggcg gcaccttcca gaggccccgg gcgcagggta gaccggggcggggcggcccg 240 cggacaggtg cagccccagg cgcaggcgca ctcgcgcctc ccggcgcaggcggtgaacct 300 cgccccaccc cagcccctcc ggggggcagc tgggccgggt cgggaggggcccaccagccc 360 gggagacact ccatatacgg ccaggcccgc tttacctggg ctccggccaggccgctcctt 420 ctttggtcag cacaggggac ccgggcgggg gcccaggccg ctaacccgccgggggagggg 480 gctccagtgc ccaacaccca aatatggctc gagaagggga gcgacattccagtgaggcgg 540 ctcgggggga gaacccgcgg gctatataaa acctgagcgt ggggaccagcggccaccgca 600 gcggacagcg ccgagagaag cctcgcttcc ctcccgcggc gaccagggccccagccggag 660 agcagcaggt gtagccacca agcttgccac catgccactc tgggtgttcttctttgtgat 720 cctcaccctc agcaacagct cccactgctc cccacctccc cctttgaccctcaggatgcg 780 gcggtattat gcagatgcca tcttcaccaa cagctaccgg aaggtgctgggccagctgtc 840 cgcccgcaag ctcctccagg acatcatgag caggtagtct agagtcggggcggccggccg 900 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaactagaatgcag 960 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgtaaccattata 1020 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttcaggttcagggg 1080 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaaaatcgataag 1140 gatccgtcga ccgatgccct tgagagcctt caacccagtc agctccttccggtgggcgcg 1200 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaactcgtaggaca 1260 ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcggtcgttcggc 1320 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccacagaatcagggg 1380 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaaccgtaaaaagg 1440 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcacaaaaatcgac 1500 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcgtttccccctg 1560 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatacctgtccgcct 1620 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtatctcagttcgg 1680 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcagcccgaccgct 1740 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgacttatcgccac 1800 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggtgctacagagt 1860 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggtatctgcgctc 1920 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggcaaacaaacca 1980 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcagaaaaaaaggat 2040 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaacgaaaactcac 2100 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatccttttaaatt 2160 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtctgacagttacc 2220 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttcatccatagttg 2280 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatctggccccagtg 2340 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagcaataaaccagc 2400 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctccatccagtcta 2460 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttgcgcaacgttg 2520 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggcttcattcagct 2580 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaaaaagcggtta 2640 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgttatcactcatgg 2700 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgcttttctgtga 2760 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccgagttgctctt 2820 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaagtgctcatca 2880 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttgagatccagtt 2940 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttcaccagcgttt 3000 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagggcgacacgga 3060 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttatcagggttatt 3120 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaataggggttccgc 3180 gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcattaagcgcggcgg 3240 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcgcccgctcctt 3300 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaagctctaaatc 3360 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgaccccaaaaaacttg 3420 attagggtga tggttcacgt agtgggccat cgccctgata gacggtttttcgccctttga 3480 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaacaacactcaacc 3540 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcctattggttaa 3600 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatattaacgtttacaa 3660 tttcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtgcgggcctctt 3720 cgctattacg ccagcccaag ctaccatgat aagtaagtaa tattaaggtacgggaggtac 3780 ttggagcggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttggttttttgtg 3840 tgaatcgata gtactaacat acgctctcca tcaaaacaaa acgaaacaaaacaaactagc 3900 aaaataggct gtccccagtg caagtgcagg tgccagaaca tttctctatcgata 3954 54 5163 DNA Artificial Sequence Description of ArtificialSequence pGHRH1-29YWTSK2014 construct 54 ggtaccgcta taggagagaaaagagctgca ctgagcaccc tccttcccct ttaaatgtca 60 acagattagg agtcagtgaatgacagcaca cctcttgcta ccttagagac caaaatttaa 120 gctactcccc ttaagctatagctagagtgc acctgccagt gtctttagtc cccactgatg 180 gaacaggacc caaggtattgaagatggaac atagttattc attcatcctc taatttaaaa 240 agctggatat gctgtacagcagaaattgac ggaacaatgt aaatcaacta taacagaaga 300 aataaaaacc tggggggaaagaagctgact atgaaacccc aggagctttc tacatgggcc 360 tggactcacc aaactctttattttgtaatg gacttctgac atttttagga agggctgtcc 420 tgatgtgggc tatagaagagggtttcacat gcttcttcaa gaggacccac actgtcccag 480 ttgctgagtc ccaccaccagatgctagtgg cagctatttg gggaacactt aggcactaca 540 aaaaaatgag tgattccattctggctcaca ccatatccct gatgtacccc ttaaagcatg 600 tcactgagtt catcacagaaaattgtttcc cctgtgcctt ccacaacaag gttagagctg 660 tccttggggc caggggaagggggcagggag tgagaagcac caactggata acctcctctg 720 acccccactc caccttaccataagtagatc caaatccttc tagaaaatta ggaaggcata 780 tccccatata tcagcgatataaatagaact gcttcagcgc tctggtagac ggtgactctc 840 caaggtggac tgggaggcagcctggccttg gctgggcatc gtcctctaaa tagaaagatg 900 aacttgttca gcctttccagaaggaaaact gctgcccagc ctacagtgca acgtccttgt 960 cttccatctg gaggaagcacgggtgacata tcatctagta agggcacctc tctgtttcca 1020 cctccaggtc gaggggtgtgacccttactt ctcagcctca agggagggac actcaacccc 1080 ccaaaaagac atgagggcgctcagctcggc ccaccgcacc ccggaccgga gccgtcaccc 1140 cccgaaattc actcccttcacaagccccca agcgcgttct ctggtgcgga ctgctccggg 1200 gccctggctt tgtgcccagcgttgtcagag ccaccgccct gagcctgtcc ccgggagccc 1260 cgcgcctcct cccaccgctccgctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320 ctgcgcgccc tcccgctgccggtggccctc tccggtgggg gtggggaccg acagggtcag 1380 ccctccggat ccggggcgctccgggtagcg gggagaagtg atcgctgggg agctggggga 1440 ggggtcgcct tcctgccctacccaggactc cgggtgcgac cgctcctcta tctctccagc 1500 ccgggcgcag ggtagaccggggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1560 cgcactcgcg cctcccggcgcaggcggtga acctcgcccc accccagccc ctccgggggg 1620 cagctgggcc gggtcgggaggggcccacca gcccgggaga cactccatat acggccaggc 1680 ccgctttacc tgggctccggccaggccgct ccttctttgg tcagcacagg ggacccgggc 1740 gggggcccag gccgctaacccgccggggga gggggctcca gtgcccaaca cccaaatatg 1800 gctcgagaag gggagcgacattccagtgag gcggctcggg gggagaaccc gcgggctata 1860 taaaacctga gcgtggggaccagcggccaa gcttgccacc atgccactct gggtgttctt 1920 ctttgtgatc ctcaccctcagcaacagctc ccactgctcc ccacctcccc ctttgaccct 1980 caggatgcgg cggtattatgcagatgccat cttcaccaac agctaccgga aggtgctggg 2040 ccagctgtcc gcccgcaagctcctccagga catcatgagc aggtagtcta gagtcggggc 2100 ggccggccgc ttcgagcagacatgataaga tacattgatg agtttggaca aaccacaact 2160 agaatgcagt gaaaaaaatgctttatttgt gaaatttgtg atgctattgc tttatttgta 2220 accattataa gctgcaataaacaagttaac aacaacaatt gcattcattt tatgtttcag 2280 gttcaggggg aggtgtgggaggttttttaa agcaagtaaa acctctacaa atgtggtaaa 2340 atcgataagg atccgtcgaccgatgccctt gagagccttc aacccagtca gctccttccg 2400 gtgggcgcgg ggcatgactatcgtcgccgc acttatgact gtcttcttta tcatgcaact 2460 cgtaggacag gtgccggcagcgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2520 tcgttcggct gcggcgagcggtatcagctc actcaaaggc ggtaatacgg ttatccacag 2580 aatcagggga taacgcaggaaagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2640 gtaaaaaggc cgcgttgctggcgtttttcc ataggctccg cccccctgac gagcatcaca 2700 aaaatcgacg ctcaagtcagaggtggcgaa acccgacagg actataaaga taccaggcgt 2760 ttccccctgg aagctccctcgtgcgctctc ctgttccgac cctgccgctt accggatacc 2820 tgtccgcctt tctcccttcgggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 2880 tcagttcggt gtaggtcgttcgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2940 ccgaccgctg cgccttatccggtaactatc gtcttgagtc caacccggta agacacgact 3000 tatcgccact ggcagcagccactggtaaca ggattagcag agcgaggtat gtaggcggtg 3060 ctacagagtt cttgaagtggtggcctaact acggctacac tagaaggaca gtatttggta 3120 tctgcgctct gctgaagccagttaccttcg gaaaaagagt tggtagctct tgatccggca 3180 aacaaaccac cgctggtagcggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3240 aaaaaggatc tcaagaagatcctttgatct tttctacggg gtctgacgct cagtggaacg 3300 aaaactcacg ttaagggattttggtcatga gattatcaaa aaggatcttc acctagatcc 3360 ttttaaatta aaaatgaagttttaaatcaa tctaaagtat atatgagtaa acttggtctg 3420 acagttacca atgcttaatcagtgaggcac ctatctcagc gatctgtcta tttcgttcat 3480 ccatagttgc ctgactccccgtcgtgtaga taactacgat acgggagggc ttaccatctg 3540 gccccagtgc tgcaatgataccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3600 taaaccagcc agccggaagggccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3660 tccagtctat taattgttgccgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3720 gcaacgttgt tgccattgctacaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3780 cattcagctc cggttcccaacgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3840 aagcggttag ctccttcggtcctccgatcg ttgtcagaag taagttggcc gcagtgttat 3900 cactcatggt tatggcagcactgcataatt ctcttactgt catgccatcc gtaagatgct 3960 tttctgtgac tggtgagtactcaaccaagt cattctgaga atagtgtatg cggcgaccga 4020 gttgctcttg cccggcgtcaatacgggata ataccgcgcc acatagcaga actttaaaag 4080 tgctcatcat tggaaaacgttcttcggggc gaaaactctc aaggatctta ccgctgttga 4140 gatccagttc gatgtaacccactcgtgcac ccaactgatc ttcagcatct tttactttca 4200 ccagcgtttc tgggtgagcaaaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4260 cgacacggaa atgttgaatactcatactct tcctttttca atattattga agcatttatc 4320 agggttattg tctcatgagcggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4380 gggttccgcg cacatttccccgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa 4440 gcgcggcggg tgtggtggttacgcgcagcg tgaccgctac acttgccagc gccctagcgc 4500 ccgctccttt cgctttcttcccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4560 ctctaaatcg ggggctccctttagggttcc gatttagtgc tttacggcac ctcgacccca 4620 aaaaacttga ttagggtgatggttcacgta gtgggccatc gccctgatag acggtttttc 4680 gccctttgac gttggagtccacgttcttta atagtggact cttgttccaa actggaacaa 4740 cactcaaccc tatctcggtctattcttttg atttataagg gattttgccg atttcggcct 4800 attggttaaa aaatgagctgatttaacaaa aatttaacgc gaattttaac aaaatattaa 4860 cgtttacaat ttcccattcgccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 4920 gggcctcttc gctattacgccagcccaagc taccatgata agtaagtaat attaaggtac 4980 gggaggtact tggagcggccgcaataaaat atctttattt tcattacatc tgtgtgttgg 5040 ttttttgtgt gaatcgatagtactaacata cgctctccat caaaacaaaa cgaaacaaaa 5100 caaactagca aaataggctgtccccagtgc aagtgcaggt gccagaacat ttctctatcg 5160 ata 5163 55 5111 DNAArtificial Sequence Description of Artificial SequencepGHRH1-29Yala1522CMV construct 55 gctgtgcctt ctagttgcca gccatctgttgtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcctaataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggtggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggatgcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc ctgggccagaaagaagcagg cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagttccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgctaaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac ctagcctccaagagtgggaa gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctccaacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactgactcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaatacggttatcc acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagcaaaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccctgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga caggactataaagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgccgcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctcacgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacgaaccccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaacccggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgaggtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct acactagaagaacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtagctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagcagattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta cggggtctgacgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggatcttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatgagtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct cagcgatctgtctatttcgt tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcgtgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagtgagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga ttttgaacttttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactcagcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgccagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat caaatgaaactgcaatttat tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaatgaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcgattccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa aataaggttatcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgcatttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgcatcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctgttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgcatcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccggggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtcggaagaggca taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattggcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaatcgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaatcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatggctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgatatatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg gctttccccccccccccatt attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaatgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacctgacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg tatcacgaggccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag agcagattgtactgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccgcatcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttatattggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatgacgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg ggtggagtatttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccctattgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgggactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaatgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgcttccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattattgaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg gctctttgccacaactatct ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactctgtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccgtcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga atctcgggtacgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggtcccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca gtggaggccagacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtagggtatgtgtc tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagacttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcagaggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcgttgctgccgc gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttccatgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg gccgctctagaccaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccctcagcaacag ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtattatgcagatgc catcttcacc aacagctacc 5040 ggaaggtgct ggcccagctg tccgcccgcaaggccctcca ggacatcatg agcaggtaga 5100 gatccagatc t 5111 56 3327 DNAArtificial Sequence Description of Artificial SequencepGHRH1-29Yala1522SK construct 56 ggtaccgagc tcttacgcgt gctagcccgggctcgagatc tgcgatctaa gtaagcttgc 60 caccatgcca ctctgggtgt tcttctttgtgatcctcacc ctcagcaaca gctcccactg 120 ctccccacct ccccctttga ccctcaggatgcggcggtat tatgcagatg ccatcttcac 180 caacagctac cggaaggtgc tggcccagctgtccgcccgc aaggccctcc aggacatcat 240 gagcaggtag tctagagtcg gggcggccggccgcttcgag cagacatgat aagatacatt 300 gatgagtttg gacaaaccac aactagaatgcagtgaaaaa aatgctttat ttgtgaaatt 360 tgtgatgcta ttgctttatt tgtaaccattataagctgca ataaacaagt taacaacaac 420 aattgcattc attttatgtt tcaggttcagggggaggtgt gggaggtttt ttaaagcaag 480 taaaacctct acaaatgtgg taaaatcgataaggatccgt cgaccgatgc ccttgagagc 540 cttcaaccca gtcagctcct tccggtgggcgcggggcatg actatcgtcg ccgcacttat 600 gactgtcttc tttatcatgc aactcgtaggacaggtgccg gcagcgctct tccgcttcct 660 cgctcactga ctcgctgcgc tcggtcgttcggctgcggcg agcggtatca gctcactcaa 720 aggcggtaat acggttatcc acagaatcaggggataacgc aggaaagaac atgtgagcaa 780 aaggccagca aaaggccagg aaccgtaaaaaggccgcgtt gctggcgttt ttccataggc 840 tccgcccccc tgacgagcat cacaaaaatcgacgctcaag tcagaggtgg cgaaacccga 900 caggactata aagataccag gcgtttccccctggaagctc cctcgtgcgc tctcctgttc 960 cgaccctgcc gcttaccgga tacctgtccgcctttctccc ttcgggaagc gtggcgcttt 1020 ctcaatgctc acgctgtagg tatctcagttcggtgtaggt cgttcgctcc aagctgggct 1080 gtgtgcacga accccccgtt cagcccgaccgctgcgcctt atccggtaac tatcgtcttg 1140 agtccaaccc ggtaagacac gacttatcgccactggcagc agccactggt aacaggatta 1200 gcagagcgag gtatgtaggc ggtgctacagagttcttgaa gtggtggcct aactacggct 1260 acactagaag gacagtattt ggtatctgcgctctgctgaa gccagttacc ttcggaaaaa 1320 gagttggtag ctcttgatcc ggcaaacaaaccaccgctgg tagcggtggt ttttttgttt 1380 gcaagcagca gattacgcgc agaaaaaaaggatctcaaga agatcctttg atcttttcta 1440 cggggtctga cgctcagtgg aacgaaaactcacgttaagg gattttggtc atgagattat 1500 caaaaaggat cttcacctag atccttttaaattaaaaatg aagttttaaa tcaatctaaa 1560 gtatatatga gtaaacttgg tctgacagttaccaatgctt aatcagtgag gcacctatct 1620 cagcgatctg tctatttcgt tcatccatagttgcctgact ccccgtcgtg tagataacta 1680 cgatacggga gggcttacca tctggccccagtgctgcaat gataccgcga gacccacgct 1740 caccggctcc agatttatca gcaataaaccagccagccgg aagggccgag cgcagaagtg 1800 gtcctgcaac tttatccgcc tccatccagtctattaattg ttgccgggaa gctagagtaa 1860 gtagttcgcc agttaatagt ttgcgcaacgttgttgccat tgctacaggc atcgtggtgt 1920 cacgctcgtc gtttggtatg gcttcattcagctccggttc ccaacgatca aggcgagtta 1980 catgatcccc catgttgtgc aaaaaagcggttagctcctt cggtcctccg atcgttgtca 2040 gaagtaagtt ggccgcagtg ttatcactcatggttatggc agcactgcat aattctctta 2100 ctgtcatgcc atccgtaaga tgcttttctgtgactggtga gtactcaacc aagtcattct 2160 gagaatagtg tatgcggcga ccgagttgctcttgcccggc gtcaatacgg gataataccg 2220 cgccacatag cagaacttta aaagtgctcatcattggaaa acgttcttcg gggcgaaaac 2280 tctcaaggat cttaccgctg ttgagatccagttcgatgta acccactcgt gcacccaact 2340 gatcttcagc atcttttact ttcaccagcgtttctgggtg agcaaaaaca ggaaggcaaa 2400 atgccgcaaa aaagggaata agggcgacacggaaatgttg aatactcata ctcttccttt 2460 ttcaatatta ttgaagcatt tatcagggttattgtctcat gagcggatac atatttgaat 2520 gtatttagaa aaataaacaa ataggggttccgcgcacatt tccccgaaaa gtgccacctg 2580 acgcgccctg tagcggcgca ttaagcgcggcgggtgtggt ggttacgcgc agcgtgaccg 2640 ctacacttgc cagcgcccta gcgcccgctcctttcgcttt cttcccttcc tttctcgcca 2700 cgttcgccgg ctttccccgt caagctctaaatcgggggct ccctttaggg ttccgattta 2760 gtgctttacg gcacctcgac cccaaaaaacttgattaggg tgatggttca cgtagtgggc 2820 catcgccctg atagacggtt tttcgccctttgacgttgga gtccacgttc tttaatagtg 2880 gactcttgtt ccaaactgga acaacactcaaccctatctc ggtctattct tttgatttat 2940 aagggatttt gccgatttcg gcctattggttaaaaaatga gctgatttaa caaaaattta 3000 acgcgaattt taacaaaata ttaacgtttacaatttccca ttcgccattc aggctgcgca 3060 actgttggga agggcgatcg gtgcgggcctcttcgctatt acgccagccc aagctaccat 3120 gataagtaag taatattaag gtacgggaggtacttggagc ggccgcaata aaatatcttt 3180 attttcatta catctgtgtg ttggttttttgtgtgaatcg atagtactaa catacgctct 3240 ccatcaaaac aaaacgaaac aaaacaaactagcaaaatag gctgtcccca gtgcaagtgc 3300 aggtgccaga acatttctct atcgata 332757 3954 DNA Artificial Sequence Description of Artificial SequencepGHRH1-29Yala1522SK construct 57 ggtaccatcg ctggggagct gggggaggggtcgccttcct gccctaccca ggactccggg 60 tgcgaccgct cctctatctc tccagcccaccaccactcca ccacttggac acgtctccct 120 cctccctgga gtcgctctag agggtttgggggtctgagta aagaacccga agtagggata 180 cagtgtggcg gcaccttcca gaggccccgggcgcagggta gaccggggcg gggcggcccg 240 cggacaggtg cagccccagg cgcaggcgcactcgcgcctc ccggcgcagg cggtgaacct 300 cgccccaccc cagcccctcc ggggggcagctgggccgggt cgggaggggc ccaccagccc 360 gggagacact ccatatacgg ccaggcccgctttacctggg ctccggccag gccgctcctt 420 ctttggtcag cacaggggac ccgggcgggggcccaggccg ctaacccgcc gggggagggg 480 gctccagtgc ccaacaccca aatatggctcgagaagggga gcgacattcc agtgaggcgg 540 ctcgggggga gaacccgcgg gctatataaaacctgagcgt ggggaccagc ggccaccgca 600 gcggacagcg ccgagagaag cctcgcttccctcccgcggc gaccagggcc ccagccggag 660 agcagcaggt gtagccacca agcttgccaccatgccactc tgggtgttct tctttgtgat 720 cctcaccctc agcaacagct cccactgctccccacctccc cctttgaccc tcaggatgcg 780 gcggtattat gcagatgcca tcttcaccaacagctaccgg aaggtgctgg cccagctgtc 840 cgcccgcaag gccctccagg acatcatgagcaggtagtct agagtcgggg cggccggccg 900 cttcgagcag acatgataag atacattgatgagtttggac aaaccacaac tagaatgcag 960 tgaaaaaaat gctttatttg tgaaatttgtgatgctattg ctttatttgt aaccattata 1020 agctgcaata aacaagttaa caacaacaattgcattcatt ttatgtttca ggttcagggg 1080 gaggtgtggg aggtttttta aagcaagtaaaacctctaca aatgtggtaa aatcgataag 1140 gatccgtcga ccgatgccct tgagagccttcaacccagtc agctccttcc ggtgggcgcg 1200 gggcatgact atcgtcgccg cacttatgactgtcttcttt atcatgcaac tcgtaggaca 1260 ggtgccggca gcgctcttcc gcttcctcgctcactgactc gctgcgctcg gtcgttcggc 1320 tgcggcgagc ggtatcagct cactcaaaggcggtaatacg gttatccaca gaatcagggg 1380 ataacgcagg aaagaacatg tgagcaaaaggccagcaaaa ggccaggaac cgtaaaaagg 1440 ccgcgttgct ggcgtttttc cataggctccgcccccctga cgagcatcac aaaaatcgac 1500 gctcaagtca gaggtggcga aacccgacaggactataaag ataccaggcg tttccccctg 1560 gaagctccct cgtgcgctct cctgttccgaccctgccgct taccggatac ctgtccgcct 1620 ttctcccttc gggaagcgtg gcgctttctcaatgctcacg ctgtaggtat ctcagttcgg 1680 tgtaggtcgt tcgctccaag ctgggctgtgtgcacgaacc ccccgttcag cccgaccgct 1740 gcgccttatc cggtaactat cgtcttgagtccaacccggt aagacacgac ttatcgccac 1800 tggcagcagc cactggtaac aggattagcagagcgaggta tgtaggcggt gctacagagt 1860 tcttgaagtg gtggcctaac tacggctacactagaaggac agtatttggt atctgcgctc 1920 tgctgaagcc agttaccttc ggaaaaagagttggtagctc ttgatccggc aaacaaacca 1980 ccgctggtag cggtggtttt tttgtttgcaagcagcagat tacgcgcaga aaaaaaggat 2040 ctcaagaaga tcctttgatc ttttctacggggtctgacgc tcagtggaac gaaaactcac 2100 gttaagggat tttggtcatg agattatcaaaaaggatctt cacctagatc cttttaaatt 2160 aaaaatgaag ttttaaatca atctaaagtatatatgagta aacttggtct gacagttacc 2220 aatgcttaat cagtgaggca cctatctcagcgatctgtct atttcgttca tccatagttg 2280 cctgactccc cgtcgtgtag ataactacgatacgggaggg cttaccatct ggccccagtg 2340 ctgcaatgat accgcgagac ccacgctcaccggctccaga tttatcagca ataaaccagc 2400 cagccggaag ggccgagcgc agaagtggtcctgcaacttt atccgcctcc atccagtcta 2460 ttaattgttg ccgggaagct agagtaagtagttcgccagt taatagtttg cgcaacgttg 2520 ttgccattgc tacaggcatc gtggtgtcacgctcgtcgtt tggtatggct tcattcagct 2580 ccggttccca acgatcaagg cgagttacatgatcccccat gttgtgcaaa aaagcggtta 2640 gctccttcgg tcctccgatc gttgtcagaagtaagttggc cgcagtgtta tcactcatgg 2700 ttatggcagc actgcataat tctcttactgtcatgccatc cgtaagatgc ttttctgtga 2760 ctggtgagta ctcaaccaag tcattctgagaatagtgtat gcggcgaccg agttgctctt 2820 gcccggcgtc aatacgggat aataccgcgccacatagcag aactttaaaa gtgctcatca 2880 ttggaaaacg ttcttcgggg cgaaaactctcaaggatctt accgctgttg agatccagtt 2940 cgatgtaacc cactcgtgca cccaactgatcttcagcatc ttttactttc accagcgttt 3000 ctgggtgagc aaaaacagga aggcaaaatgccgcaaaaaa gggaataagg gcgacacgga 3060 aatgttgaat actcatactc ttcctttttcaatattattg aagcatttat cagggttatt 3120 gtctcatgag cggatacata tttgaatgtatttagaaaaa taaacaaata ggggttccgc 3180 gcacatttcc ccgaaaagtg ccacctgacgcgccctgtag cggcgcatta agcgcggcgg 3240 gtgtggtggt tacgcgcagc gtgaccgctacacttgccag cgccctagcg cccgctcctt 3300 tcgctttctt cccttccttt ctcgccacgttcgccggctt tccccgtcaa gctctaaatc 3360 gggggctccc tttagggttc cgatttagtgctttacggca cctcgacccc aaaaaacttg 3420 attagggtga tggttcacgt agtgggccatcgccctgata gacggttttt cgccctttga 3480 cgttggagtc cacgttcttt aatagtggactcttgttcca aactggaaca acactcaacc 3540 ctatctcggt ctattctttt gatttataagggattttgcc gatttcggcc tattggttaa 3600 aaaatgagct gatttaacaa aaatttaacgcgaattttaa caaaatatta acgtttacaa 3660 tttcccattc gccattcagg ctgcgcaactgttgggaagg gcgatcggtg cgggcctctt 3720 cgctattacg ccagcccaag ctaccatgataagtaagtaa tattaaggta cgggaggtac 3780 ttggagcggc cgcaataaaa tatctttattttcattacat ctgtgtgttg gttttttgtg 3840 tgaatcgata gtactaacat acgctctccatcaaaacaaa acgaaacaaa acaaactagc 3900 aaaataggct gtccccagtg caagtgcaggtgccagaaca tttctctatc gata 3954 58 5283 DNA Artificial SequenceDescription of Artificial Sequence pGHRH1-29Yala1522SK2014 construct 58ggtaccgcta taggagagaa aagagctgca ctgagcaccc tccttcccct ttaaatgtca 60acagattagg agtcagtgaa tgacagcaca cctcttgcta ccttagagac caaaatttaa 120gctactcccc ttaagctata gctagagtgc acctgccagt gtctttagtc cccactgatg 180gaacaggacc caaggtattg aagatggaac atagttattc attcatcctc taatttaaaa 240agctggatat gctgtacagc agaaattgac ggaacaatgt aaatcaacta taacagaaga 300aataaaaacc tggggggaaa gaagctgact atgaaacccc aggagctttc tacatgggcc 360tggactcacc aaactcttta ttttgtaatg gacttctgac atttttagga agggctgtcc 420tgatgtgggc tatagaagag ggtttcacat gcttcttcaa gaggacccac actgtcccag 480ttgctgagtc ccaccaccag atgctagtgg cagctatttg gggaacactt aggcactaca 540aaaaaatgag tgattccatt ctggctcaca ccatatccct gatgtacccc ttaaagcatg 600tcactgagtt catcacagaa aattgtttcc cctgtgcctt ccacaacaag gttagagctg 660tccttggggc caggggaagg gggcagggag tgagaagcac caactggata acctcctctg 720acccccactc caccttacca taagtagatc caaatccttc tagaaaatta ggaaggcata 780tccccatata tcagcgatat aaatagaact gcttcagcgc tctggtagac ggtgactctc 840caaggtggac tgggaggcag cctggccttg gctgggcatc gtcctctaaa tagaaagatg 900aacttgttca gcctttccag aaggaaaact gctgcccagc ctacagtgca acgtccttgt 960cttccatctg gaggaagcac gggtgacata tcatctagta agggcacctc tctgtttcca 1020cctccaggtc gaggggtgtg acccttactt ctcagcctca agggagggac actcaacccc 1080ccaaaaagac atgagggcgc tcagctcggc ccaccgcacc ccggaccgga gccgtcaccc 1140cccgaaattc actcccttca caagccccca agcgcgttct ctggtgcgga ctgctccggg 1200gccctggctt tgtgcccagc gttgtcagag ccaccgccct gagcctgtcc ccgggagccc 1260cgcgcctcct cccaccgctc cgctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320ctgcgcgccc tcccgctgcc ggtggccctc tccggtgggg gtggggaccg acagggtcag 1380ccctccggat ccggggcgct ccgggtagcg gggagaagtg atcgctgggg agctggggga 1440ggggtcgcct tcctgcccta cccaggactc cgggtgcgac cgctcctcta tctctccagc 1500ccaccaccac tccaccactt ggacacgtct ccctcctccc tggagtcgct ctagagggtt 1560tgggggtctg agtaaagaac ccgaagtagg gatacagtgt ggcggcacct tccagaggcc 1620ccgggcgcag ggtagaccgg ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1680cgcactcgcg cctcccggcg caggcggtga acctcgcccc accccagccc ctccgggggg 1740cagctgggcc gggtcgggag gggcccacca gcccgggaga cactccatat acggccaggc 1800ccgctttacc tgggctccgg ccaggccgct ccttctttgg tcagcacagg ggacccgggc 1860gggggcccag gccgctaacc cgccggggga gggggctcca gtgcccaaca cccaaatatg 1920gctcgagaag gggagcgaca ttccagtgag gcggctcggg gggagaaccc gcgggctata 1980taaaacctga gcgtggggac cagcggccaa gcttgccacc atgccactct gggtgttctt 2040ctttgtgatc ctcaccctca gcaacagctc ccactgctcc ccacctcccc ctttgaccct 2100caggatgcgg cggtattatg cagatgccat cttcaccaac agctaccgga aggtgctggc 2160ccagctgtcc gcccgcaagg ccctccagga catcatgagc aggtagtcta gagtcggggc 2220ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact 2280agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 2340accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 2400gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa 2460atcgataagg atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg 2520gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact 2580cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2640tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 2700aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2760gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 2820aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 2880ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 2940tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 3000tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 3060ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3120tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3180ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 3240tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 3300aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3360aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3420aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3480ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3540acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 3600ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 3660gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3720taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3780tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3840gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3900cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3960aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 4020cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 4080tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4140gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 4200tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 4260gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 4320ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4380cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4440agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4500gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa 4560gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4620ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4680ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 4740aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 4800gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 4860cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 4920attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 4980cgtttacaat ttcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 5040gggcctcttc gctattacgc cagcccaagc taccatgata agtaagtaat attaaggtac 5100gggaggtact tggagcggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg 5160ttttttgtgt gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa 5220caaactagca aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctctatcg 5280ata 5283 59 5188 DNA Artificial Sequence Description of ArtificialSequence pGHRH1-44YWTCMV construct 59 gctgtgcctt ctagttgcca gccatctgttgtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcctaataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggtggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggatgcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc ctgggccagaaagaagcagg cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagttccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgctaaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac ctagcctccaagagtgggaa gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctccaacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactgactcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaatacggttatcc acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagcaaaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccctgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga caggactataaagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgccgcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctcacgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacgaaccccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaacccggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgaggtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct acactagaagaacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtagctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagcagattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta cggggtctgacgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggatcttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatgagtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct cagcgatctgtctatttcgt tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcgtgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagtgagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga ttttgaacttttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactcagcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgccagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat caaatgaaactgcaatttat tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaatgaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcgattccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa aataaggttatcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgcatttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgcatcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctgttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgcatcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccggggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtcggaagaggca taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattggcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaatcgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaatcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatggctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgatatatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg gctttccccccccccccatt attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaatgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacctgacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg tatcacgaggccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag agcagattgtactgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccgcatcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttatattggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatgacgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg ggtggagtatttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccctattgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgggactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaatgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgcttccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattattgaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg gctctttgccacaactatct ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactctgtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccgtcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga atctcgggtacgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggtcccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca gtggaggccagacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtagggtatgtgtc tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagacttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcagaggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcgttgctgccgc gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttccatgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg gccgctctagaccaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccctcagcaacag ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtattatgcagatgc catcttcacc aacagctacc 5040 ggaaggtgct gggccagctg tccgcccgcaagctgctcca ggacatcatg agcaggcagc 5100 agggagagag aaaccaagag caaggagcaagggtgcggct ttgaagatct tagtagtagt 5160 aggcggccgc tctagaggat ccagatct5188 60 5254 DNA Artificial Sequence Description of Artificial SequencepGHRH1-44WTGHpep construct 60 gctgtgcctt ctagttgcca gccatctgttgtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcctaataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggtggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggatgcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc ctgggccagaaagaagcagg cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagttccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgctaaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac ctagcctccaagagtgggaa gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctccaacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactgactcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaatacggttatcc acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagcaaaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccctgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga caggactataaagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgccgcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctcacgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacgaaccccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaacccggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgaggtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct acactagaagaacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtagctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagcagattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta cggggtctgacgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggatcttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatgagtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct cagcgatctgtctatttcgt tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcgtgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagtgagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga ttttgaacttttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactcagcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgccagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat caaatgaaactgcaatttat tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaatgaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcgattccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa aataaggttatcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgcatttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgcatcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctgttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgcatcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccggggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtcggaagaggca taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattggcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaatcgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaatcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatggctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgatatatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg gctttccccccccccccatt attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaatgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacctgacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg tatcacgaggccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctcccggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgcgtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag agcagattgtactgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccgcatcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttatattggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag ttattaatagtaatcaatta cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataacttacggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatgacgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg ggtggagtatttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccctattgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgggactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcggttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctccaccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaatgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtctatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgttttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcattggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact ctataggcacacccctttgg ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgcttccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattattgaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg gctctttgccacaactatct ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactctgtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccgtcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga atctcgggtacgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggtcccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca gtggaggccagacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtagggtatgtgtc tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagacttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcagaggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcgttgctgccgc gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttccatgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg gccgctctagaccaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccctcagcaacag ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtattatgcagatgc catcttcacc aacagctacc 5040 ggaaggtgct gggccagctg tccgcccgcaagctgctcca ggacatcatg agcaggcagc 5100 agggagagag aaaccaagag caaggagcaagggtgcggct tgggcggaaa gtagaaacgt 5160 ttctgcgtat tgtacagtgt cgtagcgtagaagggagctg tgggttttga agatcttagt 5220 agtagtaggc ggccgctcta gaggatccagatct 5254 61 39 DNA Artificial Sequence Description of ArtificialSequence Primer 61 agatctgcca ccatgccact ctgggtgttc ttctttgtg 39 62 36DNA Artificial Sequence Description of Artificial Sequence Primer 62ggatccaagc cgcacccttg ctccttgctc ttggtt 36 63 492 DNA ArtificialSequence Description of Artificial Sequence Primer 63 ggttttttgtggatccaagg ccgagacgta cctgcgggtc atgaagtgtc gccgcttcgt 60 ggaaagcagctgtgccttca cctacaaaga gtttgagcgg gcgtacatcc ccgagggaca 120 gaggtactccatccagaacg cgcaggccgc cttctgcttc tcggagacca tcccggcccc 180 cacgggcaaggacgaggccc agcagcgatc cgacgtggag ctgctccgct tctccctgct 240 gctcatccagtcgtggctcg ggcccgtgca gtttctcagc agggtcttca ccaacagcct 300 ggtgttcggcacctcagacc gagtctacga gaagctcaag gacctggagg aaggcatcca 360 agccctgatgcgggagctgg aagatggcag tccccgggcc gggcagatcc tgaagcagac 420 ctacgacaagtttgacacga acctgcgcag tgacgatgcg ctgcttaaga actacgggct 480 gctctcctgc tt492 64 69 DNA Artificial Sequence Description of Artificial SequencePrimer 64 ggatccgaag gcacagctgc tttccacgaa gcggcgacac ttcatgacccgcaggtacgt 60 ctcggcctt 69 65 102 DNA Artificial Sequence Description ofArtificial Sequence Primer 65 agatcttcaa agccgcaccc ttgctccttgctcttggttt ctctctccct gctgcctgct 60 catgatgtcc tggagcagct tgcgggcggacagctggccc ag 102 66 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 66 ccgcggcatc ctgagggtca a 21 67 21 DNAArtificial Sequence Description of Artificial Sequence Primer 67tatgcagatg ccatcttcaa c 21

1. A method for the treating growth hormone related disorderscharacterized by growth hormone deficiencies in an animal, comprisingsupplying the animal with a polynucleotide sequence that encodes growthhormone releasing hormone or modified growth hormone releasing hormone.2. A method for improving the growth and performance of an animal,comprising supplying the animal with a polynucleotide sequence thatencodes growth hormone releasing hormone or modified growth hormonereleasing hormone.
 3. The method of claim 1 or 2 wherein apolynucleotide sequence encoding growth hormone releasing hormone ormodified growth hormone releasing hormone is contained inpharmaceutically acceptable carrier and is administered to an animal. 4.The method of claim 3 in which the carrier is a DNA vector, a viralvector, a liposome or lipofectin.
 5. The method of claim 4 in which theDNA vector is an expression vector.
 6. The expression vector of claim 5containing a polynucleotide sequence of growth hormone releasing hormoneor modified growth hormone releasing hormone in operative associationwith a nucleotide regulatory sequence that controls expression of thepolynucleotide.
 7. The expression vector of claim 6, wherein saidregulatory element is selected from the group consisting of thecytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus, and the swine alpha-skeletal actin promoter.
 8. Themethod of claim 2 in which the animal is a cat, dog, cow, pig, horse, orchicken.
 9. A method for the treating growth hormone related disorderscharacterized by growth hormone deficiencies in an animal, comprisingsupplying the animal with a polynucleotide sequence that encodes growthhormone or modified growth hormone.
 10. A method for improving thegrowth and performance of an animal, comprising supplying the animalwith a polynucleotide sequence that encodes growth hormone or modifiedgrowth hormone.
 11. The method of claim 9 or 10 wherein a polynucleotidesequence encoding growth hormone or modified growth hormone is containedin pharmaceutically acceptable carrier and is administered to an animal.12. The method of claim 11 in which the carrier is a DNA vector, a viralvector, a liposome or lipofectin.
 13. The method of claim 12 in whichthe DNA vector is an expression vector.
 14. The method of claim 13,wherein the expression vector includes a polynucleotide sequence ofgrowth hormone or modified growth hormone in operative association witha nucleotide regulatory sequence that controls expression of thepolynucleotide.
 15. The method of claim 14, wherein the regulatoryelement is selected from the group consisting of the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, and the swine alpha-skeletal actin promoter.
 16. The methodof claim 15 in which the animal is a dog, cat, cow, pig, horse, orchicken
 17. A growth hormone releasing hormone (GHRH) variant comprisingthe addition of one amino acid to the amino terminus of a 29 amino acidamino terminal fragment of GHRH, in a pharmaceutical formulationsuitable for delivery to a human or livestock.
 18. The growth hormonereleasing hormone of claim 17 wherein the amino acid is a hydrophobicresidue or tyrosine.
 19. A growth hormone releasing hormone variantcomprising the addition of two or three amino acids to the aminoterminus, of a 29 amino acid amino terminal fragment of GHRH wherein thesecond amino acid is not proline or alanine, and in a pharmaceuticalformulation suitable for delivery to a human or livestock.
 20. Thegrowth hormone releasing hormone variant of claim 19 comprising theaddition of more than three amino acids to the amino terminus of a 29amino acid amino terminal fragment of GHRH, wherein the addition doesnot interfere with the functional activity of growth hormone lreleasinghormone.
 21. The growth hormone releasing hormone variant of claim 17,19, or 20, further comprising a substitution of glycine with alanine atresidue
 15. 22. The growth hormone releasing hormone variant of claim17, 19, or 20 further comprising a substitution of leucine with alanineat residue
 22. 23. The growth hormone releasing hormone variant of claim17, 19, or 20, further comprising substitutions of glycine with alanineat residue 15 and leucine with alanine at residue
 22. 24. The growthhormone releasing hormone variant of claim 17, 19, or 20, furthercomprising the addition of glycine and arginine at the carboxy-terminus.25. The growth hormone releasing hormone variant of claim 18, 19, or 21in which the amino acids are naturally occurring.
 26. A polynucleotidesequence encoding the growth hormone releasing hormone variant of claim17, 19, or
 20. 27. A nucleotide vector containing the polynucleotidesequence of claim
 26. 28. An expression vector containing thepolynucleotide sequence of claim 26 in operative association with anucleotide regulatory sequence that controls expression of thepolynucleotide sequence in a host cell.
 29. The expression vector ofclaim 28, wherein said regulatory element is selected from the groupconsisting of the cytomegalovirus hCMV immediate early gene, the earlyor late promoters of SV40 adenovirus, and the swine alpha-skeletal actinpromoter.
 30. A genetically engineered host cell that contains thepolynucleotide sequence of claim
 26. 31. A genetically engineered hostcell that contains the polynucleotide sequence of claim 26 in operativeassociation with a nucleotide regulatory sequence that controlsexpression of the polynucleotide sequence in the host cell.
 32. A methodfor the treating growth hormone related disorders characterized bygrowth hormone deficiencies in an animal comprising supplying the animalwith a polynucleotide sequence that encodes the growth hormone releasinghormone variant of claim 17, 19, or
 20. 33. A method for improving thegrowth and performance of an animal, comprising supplying the animalwith a polynucleotide sequence that encodes a growth hormone releasinghormone variant of claim 17, 19, or
 20. 34. A purified polypeptide ofthe growth hormone releasing hormone variant of claim 17, 19, or
 20. 35.A method for the treating growth hormone related disorders characterizedby growth hormone deficiencies in an animal, comprising supplying theanimal with an effective amount of a polypeptide of claim
 35. 36. Amethod for improving the growth and performance of an animal, comprisingsupplying the animal with an effective amount of a polypeptide of claim34.
 37. A pharmaceutical composition for promoting the expression andelevation of growth hormone in an animal, comprising administering tosaid animal an effective amount of the growth hormone releasing hormonevariant of claim 17, 19, or
 20. 38. A pharmaceutical composition for thetreatment of growth hormone related disorders characterized by growthhormone deficiencies in an animal, comprising administering to saidanimal an effective amount of the growth hormone releasing hormonevariant of claim 17, 19, or
 20. 39. A pharmaceutical composition for theimprovement of growth and performance of an animal, comprisingadministering to said animal an effective amount of a growth hormonereleasing hormone variant of claim 17, 19, or 20.